Home

SMART cDNA Library Construction Kit User Manual

image

Contents

1. F No distinct bands distinguishable in the ds cDNA product using Primer Extension Section VI B 6 Compare the ds cDNA band profile to your starting RNA ds cDNA generated using primer extension should reflect a similar profile If you observe few or no bands you will need to repeat the primer ex tension step with the original tube for 2 3 additional cycles For some mammalian RNA sources e g human brain spleen and thymus distinct bands may be absent due to high complexity of the poly A RNA In addition gel running parameters can alter distinct band vis ibility Be sure to use the following conditions for optimal visibility of your bands a 1XTAE buffer instead of 1XTBE a gel concentration of 1 196 1 296 agarose and a running voltage in the range of 60 90 V Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 39 SMART cDNA Library Construction Kit User Manual VIII Troubleshooting Guide continued G Presence of low molecular weight 0 1 kb material in the size frac tionated ds cDNA The size fractionation columns are optimized to efficiently remove low molecular weight cDNA fragments small DNA contaminants and unincorporated nucleotides from the cDNA Failure to remove low molecular weight contaminants will result in a library having a preponderance of very small inserts and or apparently nonrecombinant clones Very small inserts do not necessarily knock out lacZ function
2. Store at 20 C LB broth 10 g L Bacto tryptone 5 g L Bacto yeast extract 5 g L NaCl Adjust pH to 70 with 5 N NaOH Autoclave LB agar plates Prepare LB broth as described above Add agar 15 g L and autoclave Pour plates and store at 4 C LB tet agar plates To one liter of LB broth recipe above add 15 g agar Autoclave Cool to 50 C before adding tetracycline 15 ug ml final concentration Pour plates and store at 4 C LB kan cam agar plates To one liter of LB broth recipe above add 15 g agar Autoclave Cool to 50 C before adding kanamycin 50 ug ml final concentration and chloramphenicol 34 ug ml final concentration Pour plates and store at 4 C Transduction and titering of phage in E coli When preparing media for phage transductions or plaque titering use recipes containing 10 mM MgSO for optimal adsorption of phage to bacteria For the same reason add 0 2 maltose to the LB broth when growing overnight bacterial cultures for transduction titering MgSO 1 M stock solution Dissolve 24 65 g of MgSO 7H50 in 100 ml of deionized H O Filter sterilize 20 Maltose stock solution Dissolve 20 g of maltose in 80 ml of deionized H30 bring volume up to 100 ml Filter sterilize and store at 4 C LB MgSO agar plates To one liter of LB broth add 10 ml 1M MgSO 10 mM final concentration 15 g agar Autoclave Pour plates and store at 4 C LB MgSO broth To one liter of LB broth add 10 ml 1M MgSO 10 m
3. 10 min Collect the supernatant into another sterile 50 ml tube fasten cap tightly and place at 4 C 12 Determine the titer of the amplified library Section VII F below 13 The amplified library can be stored at 4 C for up to 6 months For long term storage up to one year make 1 ml aliquots add DMSO to a final concentration of 7 and place at 70 C Avoid repeated freeze thaw cycles FE Titering the Amplified Library 1 Picka single isolated colony from the working stock plate of XL1 Blue StepVII B 5 and use itto inoculate 20 ml of LB MgSO maltose broth without antibiotics Incubate at 37 C overnight with shaking 140 rpm until the ODgoo of the culture reaches 2 0 Centrifuge the cells at 5 000 rpm for 5 min pour off the supernatant and resuspend the pellet in 75 ml of 10mM MgSO 2 Warm and dry four LB MgSO agar plates 90 mm size as explained in the note to Section VII C 3 Prepare dilutions of phage lysate library a Pipet 10 ul of the library lysate into 1 ml of 1X lambda dilution buffer Dilution 1 1 100 b Transfer 10 ul of Dilution 1 into a second tube containing 1 ml of 1X lambda dilution buffer Dilution 2 1 10 000 4 Prepare fourtubes as described inTableV using XL1 Blue overnight culture obtained from Section VII E1 and phage Dilution 2 from Section VII E3 above Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 35 SMART cDNA Library Constructio
4. 1X lambda dilution buffer As a general guideline an appropriate dilution for an unamplified lysate is 1 5 to 1 20 Perform the following steps for each dilution of each extract you wish to plate 4 Add 1 ul of the diluted phage to 200 ul of the XL1 Blue overnight culture and allow the phage to adsorb at 37 C for 10 15 min 5 Add 2 ml of melted LB MgSO top agar Mix by quickly inverting and immediately pour onto 90 mm LB MgSO plates prewarmed to 37 C Swirl the plates quickly after pouring to allow even distri bution of the top agar 6 Cool the plates at room temperature for 10 min to allow the top Clontech Laboratories Inc www clontech com Protocol No PT3000 1 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VII SMART cDNA Library Protocols continued agar to harden Invert the plates and incubate them at 37 C for 6 18 hr Periodically check the plates to ensure that plaques are developing 7 Count the plaques and calculate the titer of the phage pfu ml pfu ml number of plaques x dilution factor x 10 ul ml ul of diluted phage plated 8 Ifyou aretitering the three test ligations from SectionVII A compare the titers to determine the optimal ratio of vector arms to cDNA insert If you obtained 1 2 x 109 plaques clones altogether you may wish to repeat the ligation using the optimal ratio of vector to insert See Section VII A 6 and VIII G D Determining the Percentage of Rec
5. A for most mammalian RNA sources there should be several bands distinguishable against the background smear when a sample ofthe PCR productis runon a gel If bands are expected but are not visible and the background smear is very intense this is indicative of PCR overcycling If you suspect overcycling then the PCR step Section V B must be repeated de novo with a fresh 2 ul sample of first strand cDNA using 2 3 fewer cycles In addition gel running parameters can alter bright band visibility Be sure to usethe following conditions for optimal visibility of your bands a 1XTAE buffer instead of 1X TBE a gel concentration of 1 196 1 296 agarose and a running voltage in the range of 60 90 V E Presence of low molecular weight 0 1 kb material in the ds cDNA product using LD PCR Section V B 6 The raw cDNA e g before size fractionation is expected to contain some low molecular weight DNA contaminants including unincorpo rated primers SMART oligonucleotides and very short PCR products see Figure 6 Appendix A However these small fragments are gener ally removed from the ds cDNA preparation in the size fractionation step using the columns provided Note that a preponderance of low molecular weight 0 1 kb material in the raw PCR product may be indicative of overcycling If you suspect overcycling then the PCR step Section V B must be repeated de novo with a fresh 2 ul sample of first strand cDNA using 2 3 fewer cycles
6. For a quick analysis of o uo Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 45 SMART cDNA Library Construction Kit User Manual Appendix B Converting XTriplEx2 to pTriplEx2 continued insert size the alkaline lysis method of Birnboim amp Doly 1979 is recommended for isolation of bacterial plasmids The isolated plasmid DNA should be pure enough for direct sequencing The pTriplEx2 sequencing primers provided may be used with standard ds DNA sequencing protocols Clontech Laboratories Inc www clontech com Protocol No PT3000 1 46 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Appendix B Converting ATriplEx2 to pTriplEx2 continued ATriplEx2 42 3 kb XN a 2 Right Arm 16 8 kb Ii S pTriplEx2 A Left Arm 25 5 kb Directionally Clone cDNA into Sfi A amp B site of ATriplEx loxP loxP I l insert l N Sfi lA SfilB High efficiency single step conversion via site specific recombination at oxP sites in E coli BM25 8 Plac MCS with insert lacZ pTriplEx2 3589 bp fl ori Figure 7 Conversion of a recombinant AXTriplEx2 to the corresponding pTriplEx2 The XTriplEx2 MCS is located within an embedded plasmid which is flanked by loxP sites at the X junctions Transduction of a ATriplEx2 lysate into E coli strain BM25 8 promotes Cre recombinase medi
7. components in the reaction tube 2 ul First Strand cDNA from Step A 10 80 ul Deionized H O 10 ul 10X Advantage 2 PCR Buffer 2 ul 50X dNTP Mix 2 ul 5 PCR Primer 2 ul CDS III 3 PCR Primer 2 ul 50X Advantage 2 Polymerase Mix 100 ul Total volume 3 Mix contents by gently flicking the tube Centrifuge briefly to col lect the contents at the bottom of the tube 4 Overlay the reaction mixture with 2 drops of mineral oil if necessary Cap the tube and place it in a preheated 95 C thermal cycler 5 Commence thermal cycling using one of the following pro grams GeneAmp 480 GeneAmp 2400 9600 e 95 C 1 min e 95 C 20 sec x cycles x cycles 95 C 15sec 95 C 5sec 68 C 6 min 68 C 6min Refer to Table Ill to determine the optimal number of cycles to use 6 When the cycling is complete analyze a 5 ul sample of the PCR product alongside 0 1 ug of 1 kb DNA size markers on a 1 196 agarose EtBr gel The ds cDNA should appear as a 0 1 4 kb smear on the gel with some distinct bands corresponding to the abun dant mRNAs for that tissue or cell source CDNA prepared from some tissues may not have distinct distinct bands especially if the mRNA is highly complex If your PCR product does not appear as expected refer to the Troubleshooting Guide Section VIII A E Typical results obtained with Human Placenta Poly A RNA are Clontech Laboratories Inc www clontech com Protocol No PT3000 1 20 Version No PR7Y2399 S
8. containing this reagent If possible handle solutions containing phenol and or chloroform under a chemical fume hood B Preparation and Handling of Total and Poly At RNA 1 To avoid contamination and degradation of RNA and to minimize the presence of RNases use the following precautions a Wear gloves b Use freshly deionized e g Milli O grade H O without treat ment with DEPC diethyl pyrocarbonate c Rinse all glassware with 0 5 N NaOH followed by deionized H5O Then bake the glassware at 160 180 C for 4 9 hr d Use only single use plastic pipettes and pipette tips with RNA e Avoid using autoclaved HO because recycled steam in some autoclaves can introduce contaminants that may interfere with PCR Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 15 SMART cDNA Library Construction Kit User Manual IV General Considerations continued 2 RNA isolation Many procedures are available for the isolation of total RNA and poly A RNA Chomczynski amp Sacchi 1987 Farrell 1993 Sambrook etal 1989 Clontech offers several kits for the isolation of total RNA and subsequent isolation of poly At RNA see Section X Related Products 3 RNA analysis The sequence complexity of the ds cDNA synthesized and ulti mately of the cDNA library constructed depend on the quality of the experimental RNA starting material Therefore before you use it in a first strand synth
9. for tips on troubleshooting the size fractionation procedure Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 2 SMART cDNA Library Construction Kit User Manual IX References Barnes W M 1994 PCR amplification of up to 35 kb DNA with high fidelity and high yield from X bacteriophage templates Proc Natl Acad Sci USA 91 2216 2220 Birnboim H C amp Doly J 1979 A rapid alkaline extraction procedure for screening recombi nant plasmid DNA Nucleic Acids Res 7 1513 Borson N D Sato W L amp Drewes L R 1992 A lock docking oligo dT primer for 5 and 3 RACE PCR PCR Methods amp Appl 2 144 148 Chenchik A Diatchenko L Chang C amp Kuchibhatla S January 1994 Great Lengths cDNA Synthesis Kit for high yields of full length cDNA Clontechniques IX 1 9 12 Chenchik A Zhu Y Y Diatchenko L Li R Hill J amp Siebert P D 1998 Generation and use of high quality cDNA from small amounts of total RNA by SMART PCR In Gene Cloning and Analysis by RT PCR BioTechniques Books MA pp 305 319 Chomczynski P amp Sacchi N 1987 Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction Anal Biochem 162 156 159 D Alessio J M amp Gerard G F 1988 Second strand cDNA synthesis with E coli DNA poly merase and RNase H the fate of information at the mRNA 5 terminus and the effect of E coli DNA
10. fractions in tubes 41 16 approximately 35 ul per tube Cap each tube after each fraction is collected Recap the column after fraction 16 has been collected Checkthe profile ofthe fractions before proceeding with the experi ment Ona 1 196 agarose EtBr gel electrophorese3 ul of each fraction separately in adjacent wells alongside a 1 kb DNA size marker 0 1 ug Run the gel at 150 V for 10 min Running the gel longer will make it difficult to see the cDNA bands Determine the peak fractions by visualizing the intensity of the bands under UV Collect the first three fractions containing cDNA in most cases the fourth fraction containing cDNA is usable Make sure the fourth fraction matches your desired size distribution Pool the above fractions in a clean 1 5 ml tube Clontech Laboratories Inc www clontech com Protocol No PT3000 1 28 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VI SMART cDNA Synthesis by Primer Extension continued 11 Add the following reagents to the tube with 3 4 pooled fractions containing the cDNA 105 140 ul respectively 1 10 vol Sodium Acetate 3 M pH 4 8 1 3 ul Glycogen 20 mg ml 2 5 vol 95 ethanol 20 C 12 Mix by gently rocking the tube back and forth 13 Place the tube in 20 C or a dry ice ethanol bath for 1 hr Optional you may incubate at 20 C overnight which may result in better recovery 14 Centrifuge the tube at 14 000 rpm for 20 min at r
11. ligase Nucleic Acids Res 16 1999 2014 Farrell Jr R E 1993 RNA Methodologies A Lab Guide for Isolation and Characterization Academic Press San Diego CA Fromomt Racine M Bertrand E Pictet R amp Grange T 1993 A highly sensitive method for mapping the 5 termini of mRNAs Nucleic Acids Res 21 1683 1684 Furuichi Y amp Miura K 1975 A blocked structure at the 5 terminus of mRNA from cytoplasmic polyhedrosis virus Nature 253 374 375 Gubler U amp Hoffman B J 1983 A simple and very efficient method for generating comple mentary DNA libraries Gene 25 263 269 Huynh T V Young R A amp Davis R W 1985 DNA Cloning A Practical Approach ed Glover D M IRL Press Oxford Kato S Sekine S Oh S W Kim N S Umezawa Y Abe N Yokoyama Kobayashi M amp Aoki T 1994 Construction of a human full length cDNA bank Gene 150 243 250 Kellogg D E Rybalkin l Chen S Mukhamedova N Vlasik T Siebert P amp Chenchik A 1994 TaqStart Antibody Hot start PCR facilitated by a neutralizing monoclonal antibody directed againstTaq DNA polymerase BioTechniques 16 1134 1137 Okayama H amp Berg P 1982 High efficiency cloning of full length cDNA Mol Cell Biol 2 161 170 Sambrook J Fritsch E F amp Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor NY SMART cDNA Libra
12. should be approximately 40 ul If the flow rate is too slow i e more than 1 drop 100 sec and the volume of one drop is too small i e less than 25 ul you should resuspend the matrix completely and repeat the drip procedure until it reaches the above parameters When the storage buffer stops dripping out carefully and gently along the column inner wall add 700 ul of column buffer to the top of the column and allow it to drain out When this buffer stops dripping 15 20 min carefully and evenly apply 100 ul mixture of Sfil digested cDNA and xylene cyanol dye Section VI D 3 above to the top center surface of the matrix An unsmooth matrix surface does not hurt the following fractionation process Before proceeding to the next step allow the sample to be fully absorbed into the surface of the matrix i e there should be no liquid remaining above the surface With 100 ul of column buffer wash the tube that contained the cDNA and gently apply this material to the surface of the matrix Allow the buffer to drain out of the column until there is no liquid left above the resin When the dripping has ceased proceed to the next step At this point the dye layer should be several mm into the column Placetherackcontainingthe collection tubes StepVI E 1 underthe column so that the first tube is directly under the column outlet Add 600 ul of column buffer and immediately begin collecting single drop
13. up to 2 weeks To prepare a working stock plate pick a single isolated colony from the primary streak plate and streak it onto another LB MgSO agar plate with antibiotics Incubate at 37 C overnight Wrap plate in Parafilm and store at 4 C for up to 2 weeks This plate is used as a source of fresh colonies for inoculating liquid cultures and for preparing the next fresh working stock plate At 2 week intervals prepare a fresh working stock plate from the previous working stock plate so you will always have a source of fresh colonies Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 31 SMART cDNA Library Construction Kit User Manual VII SMART cDNA Library Protocols continued 6 If you suspect contamination on your current working stock plate prepare a new primary streak plate from the frozen culture C Titering the Unamplified Library Determining the titer e g pfu ul of the unamplified library will give you an estimate of the number of independent phage and indepen dent clones in the library A library having at least 1 x 109 independent clones in most cases is representative of the mRNA complexity The following plating titering protocol can be used to determine 1 the efficiency of the ligation of vector to positive control insert and 2 the background titer of the vector alone Notes When plating bacteria phage mixtures using melted top agar the mel
14. up to 3 months Forthe first strand synthesis and the primer extension steps all com ponents and reaction vessels should be pre chilled on ice A First Strand cDNA Synthesis 1 Combine the following reagents in a sterile 0 5 ml microcentrifuge tube 1 ul poly At RNA 1 0 ug For the control reaction use 1 ul 1 ug of the control RNA 1 ul SMART IV Oligonucleotide 1 ul CDS III 3 PCR Primer 2 pl Deionized H5O 5 ul Total volume 2 Mix contents and spin the tube briefly in a microcentrifuge 3 Incubate the tube at 72 C for 2 min 4 Cool the tube on ice for 2 min 5 Spin the tube briefly to collect the contents at the bottom Clontech Laboratories Inc www clontech com Protocol No PT3000 1 24 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VI SMART cDNA Synthesis by Primer Extension continued 6 Add the following to each reaction tube 2 0 ul 5X First Strand Buffer 1 0 ul DTT 20 mM 1 0 ul dNTP Mix 10 mM 1 0 ul MMLV ReverseTranscriptase 10 0 ul Total volume Please see Addendum PT3980 4 for details on the choice of RT enzyme 7 Mix the contents of the tube by gently pipetting and briefly spin the tube 8 Incubate the tube at 42 C for 1 hr in an air incubator or hot lid thermal cycler Note If you use a water bath or thermal cycler for this incubation cover the reaction mixture with one drop of mineral oil before you close the tube This will prevent loss of volume due t
15. 1 x 10 pfu ug of control X DNA If the unamplified library has a titer 109 pfu ug DNA all three liga tions combined but the control packaging reaction using ligated nonrecombinant X DNA yields at least 5 x 108 pfu ug there may be a problem with the ligation of vector to insert Section VII A The ligation reaction cannot be checked retroactively because the entire ligation mixture is generally used in the packaging reaction However before you repeat the ligations check the concentration of the size fraction Clontech Laboratories Inc www clontech com Protocol No PT3000 1 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VIII Troubleshooting Guide continued ated cDNA by electrophoresing 1 ul of the resuspended cDNA on an agarose EtBr gel next to a known amount of control DNA View the gel under UV light Alternatively spot 1 ul of the cDNA on an agarose EtBr plate next to small spots of known amounts 10 1 000 ng of control DNA The concentration of the resuspended cDNA should be in the range of 100 200 ng ul Checkto make sure the concentration of your ds cDNA productis within 2 3 ug before proceeding to the subsequent cloning procedure Too much starting cDNA 23 4 ug often yields poor cloning efficiencies or low titer libraries If the cDNA concentration is too high apply 5 ul of ds cDNA product to a DNA fluorometer and determine the DNA concentration Alternatively the control RNA can prov
16. ATriplEx2 Ligation Fractionation amp Ligation into ATriplex2 arms Y SfilA SfilB Packaging v cDNA library in TriplEx2 Figure 1 Flow chart of the SMART cDNA Library Construction Kit protocols The right side of the flow chart shows the fate o f incomplete transcripts caused by RNA degradation or premature termination of reverse transcription Protocol No PT3000 1 Version No PR7Y2399 Ww ww clontech com Clontech Laboratories Inc 5 SMART cDNA Library Construction Kit User Manual Introduction continued cDNA synthesis by Long Distance PCR In the first protocol Section V cDNA synthesis employs long distance PCR LD PCR Barnes et al 1994 Cheng et al 1994 for generating full length cDNA This protocol is ideal for researchers who are limited by the amount of their available RNA starting material i e 50 ng of total RNA A modified oligo dT primer CDS III 3 PCR Primer primes the first strand synthesis reaction and the SMART IV Oligo serves as a short extended template at the 5 end of the mRNA Figure 1 When the RT reaches the 5 end the enzyme s terminal transferase activity adds a few additional nucleotides primarily deoxycytidine to the 3 end of the cDNA The SMART IV Oligo which has an oligo G sequence at its 3 end base pairs with the deoxy cytidine stretch creating an extended template RT then switches templates and continues replicating to the end of the oligonucleot
17. Clontech is aTakara Bio Company 2007 Clontech Laboratories Inc Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 49
18. Ex2 cam 34 ug ml from XTriplEx2 Note For phage transductions including plaque titering library plating and screening use the following media additives for optimal adsorption of phage to bacteria e 10 mM MgSO in LB agar and LB top agar e 10mMMgSO andO 29 6 maltosein LB broth when growing overnight bacterial cultures for X phage transductions Before using XL1 Blue overnight cultures in phage transductions centrifuge the cells pour off the supernatant and resuspend the pellet in 10 mM MgSO in H O e Before using BM25 8 overnight cultures in phage transductions add MgCl to a final concentration of 10 mM e fthebacterialstrains are always maintained on stock plates contain ing the appropriate antibiotic there is no need to add antibiotics to the LB broth when growing overnight cultures Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 11 SMART cDNA Library Construction Kit User Manual Ill Additional Materials Required The following reagents are required but not supplied Store all reagents and solutions at room temperature 20 22 C unless specified otherwise First strand cDNA synthesis and SMART PCR cDNA amplification MMLV Reverse Transcriptase Please see Addendum PT3980 4 for details on the choice of RT enzyme e Advantage 2 PCR Kit Cat Nos 639206 amp 639207 Sterile 0 5 ml microcentrifuge tubes Poly A or total RNA Mineral oil We recommen
19. M 5 ul Control Insert Sfil A amp B 50 ng ul Host cells 0 5 ml E coli BM25 8 in 25 glycerol genotype in Table 0 5 ml E coli XL1 Blue in 25 glycerol genotype in Table 60 yl 5 Sequencing Primer 20 uM 60 yl 3 Sequencing Primer 20 uM General reagents 20 ul dNTP mix dATP dCTP dGTP dTTP 10 mM each e 200 yl Sodium Acetate 3 M pH 4 8 20 ul Sodium Hydroxide 25 mM 60 ul Glycogen 20 ug ul e 5 ml Deionized H O Milli O filtered not DEPC treated Clontech Laboratories Inc www clontech com Protocol No PT3000 1 10 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual ll List of Components continued TABLE I BACTERIAL HOST STRAIN GENOTYPES Strain Genotype XL1 Blue endA1 gyrA96 hsdR17 lac recA1 relA1 supE44 thi 1 F lacl 9Z AM15 proAB Tn10 Note Tn10 confers resistance to tetracycline Reference Wood et al 1985 BM25 8 supE44 thi A lac proAB F traD36 proAB lacl Z AM15 imm434 kan P1 cam hsdR r12 My27 Note BM25 8 is lysogenic for phages X and P1 and is used for automatic subcloning Reference Palazzolo et al 1990 TABLE Il HOST STRAIN APPLICATIONS amp MEDIA ADDITIVES Host Stock Application s strain plate XL1 Blue LB tet Library plating amp screening 15 ug ml e Blue white B galactosidase screening Regulated expression of cloned genes BM25 8 LB kan 50 ug ml Cre ox mediated excision of pTripl
20. M final concentration Autoclave Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 13 SMART cDNA Library Construction Kit User Manual Ill Additional Materials Required continued LB MgSO maltose broth Prepare 1 L LB MgSO broth as described above After autoclav ing cool to 50 C before adding maltose to a final concentration of 0 2 10 ml of 20 maltose stock solution LB MgSO soft top agar To one liter of LB broth add 10 ml 1M MgSO 10 mM final concentration 72 g agar Autoclave and store at 4 C 10X Lambda dilution buffer Final Conc To prepare 1 L of solution NaCl 1 0 M 58 3 g MgSO 7H 0 0 1 M 24 65 g Tris HCI pH 7 5 0 35 M 350 0 ml of 1 M Add H50 to a final volume of 1 L Autoclave and store at 4 C 1X Lambda dilution buffer 100 ml 10X Lambda dilution buffer 5 ml 296 Gelatin 0 0196 final concentration Add H 0 to a final volume of 1 L Autoclave and store at 4 C Note The 0 01 gelatin in the 1X lambda dilution buffer stabilizes the library titer for long term storage Gelatin addition is optional when diluting the phage for immediate titering Amplifying a X library LB MgSO plates LB MgSO soft top agar e 50 ml polypropylene sterile screw cap tubes Chloroform Blue white screening in E coli i e p galactosidase e IPTG 100 mM in HO Isopropyl B D thiogalactopyranoside Filter sterilize Store at 4 C e X Gal 100 mM Dissolve in dim
21. MART cDNA Library Construction Kit User Manual V SMART cDNA Synthesis by LD PCR continued 7 shown in Appendix A Proceed to the next step or store ds cDNA at 20 C until use C Proteinase K digestion oR WN Oo 12 13 14 15 In a sterile 0 5 ml tube pipet 50 ul of amplified ds cDNA 2 3 ug making sure you pipet below the top oil layer of the PCR tube and add 2 ul of proteinase K 20 ug yul Store the remaining ds cDNA at 20 C up to 3 months Note Proteinase K treatment is necessary to inactivate the DNA polymerase ac tivity This system was optimized for 2 3 ug 50 ul vol of PCR cDNA product for subsequent cloning and library construction procedures Too much ds cDNA 23 4 ug will yield a low titer library Refer to the Troubleshooting Guide Section VIII H for more information Mix contents and spin the tube briefly Incubate at 45 C for 20 min Spin the tube briefly Add 50 ul of Deionized H O to the tube Add 100 ul of phenol chloroform isoamyl alcohol and mix by con tinuous gentle inversion for 1 2 min Centrifuge at 14 000 rpm for 5 min to separate the phases Move the top aqueous layer to a clean 0 5 ml tube Discard the interface and lower layers Add 100 ul of chloroform isoamyl alcohol to the aqueous layer Mix by continuous gentle inversion for 1 2 min Centrifuge at 14 000 rpm for 5 min to separate the phases Move the top aqueous layer to a cle
22. PLEASE READ ENTIRE PROTOCOL BEFORE STARTING Important This protocol is designed for standard library construction of SMART cDNA libraries using primer extension The optimal amount of starting material for cDNA synthesis is about 1 ug of poly A RNA 0 5 ug 2 0 ug Reduced cloning efficiency will occur if you use less than 0 5 ug or greater than 2 0 ug of poly A RNA To construct SMART cDNA libraries using a PCR based method consult the protocol in Section V We strongly recommend that you perform a positive control cDNA synthe sis with the Human Placenta Poly A RNA provided in parallel with your experimental cDNA synthesis Performing a control at least once will verify that all the components are working properly This is especially important if you have difficulty visualizing your first strand reaction product on a gel Furthermore performing the control reaction in parallel with your experi ment will allow you to evaluate the yield and size distribution ofthe ds cDNA synthesized from your RNA sample Performing the control through the library construction steps will also help you troubleshoot any problems as they arise Notes e f necessary the procedure can be stopped after the first strand syn thesis by placing the reaction mixture at 20 C and at any ethanol precipitation step after the precipitated DNA has been centrifuged or when the supernatant has been removed The dry or resuspended DNA pellet can be stored at 20 C for
23. R7Y2399 SMART cDNA Library Construction Kit User Manual Il List of Components Store CHROMA SPIN Columns 1X Column Buffer and Deionized H O at room temperature Store BM25 8 and XL 1 Blue E coli host strains Control RNA and SMART IV Oligonucleotide at 70 C Store all other reagents at 20 C First strand synthesis 10 ul SMART IV Oligonucleotide 12 uM 5 AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG 3 25 ul CDS III 3 PCR Primer 12 uM 5 ATTCTAGAGGCCGAGGCGGCCGACATG d T 3gN_N 3 N A G C orT N A G or C 30 ul 5X First Strand Buffer 250 mM Tris pH 8 3 30 mM MgCl 375 mM KCI e 20 yl DTT dithiothreitol 20 mM 5 ul Control Poly At RNA Human Placenta 1 0 ug ul cDNA amplification 20 ul 5 PCR Primer 12 uM 5 AAGCAGTGGTATCAACGCAGAGT 3 Digestion of PCR enzymes 20 yl Proteinase K 20 ug ul Sfil digestion 80 yl Sfil Enzyme 20 units ul 80 ul 10X Sfil Buffer 10 ul 100X BSA cDNA purification 10 CHROMA SPIN 400 Columns 30 ml 1X Fractionation Column Buffer Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 9 SMART cDNA Library Construction Kit User Manual Il List of Components continued Vector ligation 30 yl ATripIEx2 Sfil A amp B digested arms 0 5 ug ul e 20 ul T4 DNA Ligase 400 units ul 30 ul 10X DNA Ligation Buffer 500 mM Tris HCI pH 7 8 100 mM MgCl 100 mM DTT 0 5 mg ml BSA e 20 yl ATP 10 m
24. SMART cDNA Library Construction Kit User Manual Visit our website for more details click here Cat No 634901 PT3000 1 PR7Y2399 Published 13 November 2007 SMART cDNA Library Construction Kit User Manual Table of Contents I Introduction 4 ll List of Components 9 Ill Additional Materials Required 12 IV General Considerations 15 V SMART cDNA Synthesis by LD PCR 18 A First Strand cDNA Synthesis 18 B cDNA Amplification by LD PCR 19 C Proteinase K Digestion 21 D Sfil Digestion 21 E cDNA Size Fractionation 22 VI SMART cDNA Synthesis by Primer Extension 24 A First Strand cDNA Synthesis 24 B ds cDNA Synthesis by Primer Extension 25 C Proteinase K Digestion 26 D Sfil Digestion 27 E cDNA Size Fractionation 27 VII SMART cDNA Library Protocols 30 A Ligation of cDNA to Vector 30 B Bacterial Culture Plating 31 C Titering the Unamplified Library 32 D Determining the Percentage of Recombinant Clones 33 E Library Amplification 34 EF Titering the Amplified Library 35 VIII Troubleshooting Guide 37 IX References 42 Appendix A Typical Results of PCR amp cDNA Synthesis 43 Appendix B Converting ATripIEx2 to pTriplEx2 45 Appendix C Restriction Map of TriplEx2 48 Clontech Laboratories Inc www clontech com Protocol No PT3000 1 2 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual List of Tables Table I Bacterial host strain genotypes 11 Table Il Host strain applications am
25. To avoid this be sure to check your column fractions on an agarose gel and pool only the first three fractions containing cDNA signal If you must repeat the ds cDNA synthesis and the size fractionation steps keep these points in mind 1 The resolving function of the column will be diminished if the protocol is not followed exactly In particular using too much ortoo little column buffer in washing and elution steps or omitting a step is likely to cause suboptimal results 2 Do not allow the matrix to dry out between washes or steps A dry matrix body may shrink away from the inner wall of the col umn casing The ds cDNA mixture can then flow down the sides of the column allowing small contaminants to enter the body of the matrix too quickly and elute in earlier fractions 3 The column should be stored and used at room temperature If itis chilled at 4 C and then warmed to room temperature for use bubbles may form which interfere with the proper functioning of the column 4 Extreme uneven deposition of the ds cDNA mixture on the surface of the column can cause inefficient separation of ds cDNA from low molecular weight contaminants However a slight uneven surface using the drip method is common and does not effect the resolution of the column H Low titer of unamplified library Checkto make sure you are using aA phage packaging system according to the manufacturer s specifications which is expected to yield at least
26. an 0 5 ml tube Discard the interface and lower layers Add 10 ul of 3 M Sodium Acetate 1 3 ul of Glycogen 20 ug ul and 260 ul of room temperature 9596 ethanol Immediately centrifuge at 14 000 rpm for 20 min at room temperature Note Do not chill the tube at 20 C or on ice before centrifuging Chilling the sample will result in coprecipitation of impurities Carefully remove the supernatant with a pipette Do not disturb the pellet Wash pellet with 100 ul of 8096 ethanol Air dry the pellet 210 min to evaporate off residual ethanol Add 79 ul of Deionized H O to resuspend the pellet D Sfil digestion 1 Combine the following components in a fresh 0 5 ml tube 79 ul cDNA Section V C 15 above 10 ul 10X Sfi Buffer 10 ul Sfil Enzyme 1pgl 100X BSA 100 ul Total volume Protocol No PT3000 1 www clontech com Clontech Laboratories Inc 21 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual V SMART cDNA Synthesis by LD PCR continued E 2 Mix well Incubate the tube at 50 C for 2 hr 3 Add 2 ul of 1 xylene cyanol dye to the tube above Mix well cDNA size fractionation by CHROMA SPIN 400 Label sixteen 1 5 ml tubes and arrange them in a rack in order Prepare the CHROMA SPIN 400 Column for drip procedure a Invert the column several times to completely resuspend the gel matrix b Remove air bubbles from the column Use a 1000 ul pipettor to resuspend the m
27. at is not used right away should be placed at 20 C First strand cDNA can be stored at 20 C for up to three months oan WN 1 B cDNA Amplification by LD PCR Table lll shows the relationship between the optimal number of thermal cycles to use for a given amount of RNA starting material used in the first strand synthesis Use the least number of cycles shown for that amount of RNA Fewer cycles generally mean fewer nonspecific PCR products Furthermore undercycling can be easily rectified by placing the PCR reaction back in the thermal cycler for a few more cycles see the Troubleshooting Guide Section VIII C On the other hand overcy cling requires repeating the PCR de novo using a fresh aliquot of the first strand reaction product The optimal cycling parameters in Table Ill were determined using the Control Poly A Human Placenta RNA these parameters may vary with different templates and thermal cyclers Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 19 SMART cDNA Library Construction Kit User Manual V SMART cDNA Synthesis by LD PCR continued 1 Preheat the PCR thermal cycler to 95 C TABLE Ill RELATIONSHIP BETWEEN AMOUNT OF RNA STARTING MATERIAL AND OPTIMAL NUMBER OF THERMAL CYCLES Total RNA Poly At RNA ug ug Number of Cycles 1 0 2 0 0 5 1 0 18 20 0 5 1 0 0 25 0 5 20 22 0 25 0 5 0 125 0 25 22 24 0 05 0 25 0 025 0 125 24 26 2 Combine the following
28. ated release and circularization of pTriplEx2 at the loxP sites pTriplEx2 carries the bla gene for ampicillin resistance and the pUC ori for autonomous replication in E coli The MCS provides several unique restriction sites flanking the Sfil A amp B sites to facilitate the subcloning and analysis of inserts See Appendix C for a restriction map of XTriplEx2 Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 7 SMART cDNA Library Construction Kit User Manual Appendix C Restriction Map of A TriplEx2 Multiple cutters 42 3 kb BamHI 36 0 Bgl II 33 0 amp 33 1 BamHI 29 8 Kpnl Bgl Il SfilA SfilB BamHI Kpnl 18 6 Kpnl 17 1 BamHI 5 5 Bgl II 0 42 Figure 8 Restriction map of ATriplEx2 Clontech Laboratories Inc Right arm 16 8 kb mEm MCS Left arm 25 5 kb Okb www clontech com 26 1 EcoRI Xbal Hindlll Notl Sacl 26 0 BsiWI 19 3 SnaBl 12 2 Protocol No PT3000 1 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Notes Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufact
29. atrix gently avoid generating air bubbles Then remove the bottom cap and let the column drip naturally If the column does not drain after 3 min recap the top cap This pressure should cause the column to drain c Attach the column to a ring stand Let the storage buffer drain through the column by gravity flow until you can see the surface of the gel beads in the column ma trix The top of the column matrix should be at the 1 0 ml mark on the wall of the column If your column contains significantly less matrix adjust the volume of the matrix to the 1 0 ml mark using matrix from another column an extra column is provided for this purpose e The flow rate should be approximately 1 drop 40 60 sec The volume of 1 drop should be approximately 40 ul If the flow rate istoo slow i e morethan 1 drop 100 sec and the volume of one drop is too small i e less than 25 ul you should resuspend the matrix completely and repeat the drip procedure until it reaches the above parameters a When the storage buffer stops dripping out carefully and gently along the column inner wall add 700 ul of column buffer to the top of the column and allow it to drain out When this buffer stops dripping 15 20 min carefully and evenly apply 100 ul mixture of Sfil digested cDNA and xylene cyanol dye Section V D 3 above to the top center surface of the matrix An unsmooth matrix surface does not hurt the following fractionation proc
30. clone X TriplEx2 contains two translation start sites i e two sets of ribosome binding sites and ATG start codons in different reading frames and a slip site a stretch of dTs that can cause ribosomes to shift frames between the regulated ac promoter and the MCS RNA polymerase may also slip during transcription of the dT 3 region By the time the ribosomes begin translating the insert roughly one third will be in each of the three reading frames Benefits of X TriplEx2 Vector Clontech s ATriplEx2 Vector provides all of the following advantages for cloning in a phagemid vector high titer libraries blue white screening for recombinants regulated expression of cloned inserts and ease of converting clones from phage to a plasmid vector via Cre ox mediated subcloning In addition every cDNA inserted into the MCS of XTriplEx2 is expressed in all three reading frames Figure 3 SMART IV maximizes library utility The design of the SMART IV oligonucleotide ensures that libraries made with this kit are also compatible with the Creator Cloning and Expression System The Creator System unites our wide array of expression products providing access to many strategies for gene function analysis SMART cDNA libraries can be cloned into our Creator library vector pDNR LIB Cat No 631611 via the same Sfil cloning strategy as is used with ATriplEx2 Clontech Laboratories Inc www clontech com Protocol No PT3000 1 Version No P
31. d Sigma M 3516 Perkin Elmer DNA Thermal Cyclers Note The cycling parameters in this protocol have been optimized using Applied Biosystems GeneAmp DNA Thermal Cyclers 480 2400 and 9600 along with the reagents and control RNA provided in the SMART Kit Optimal cycling parameters may vary with different templates and thermal cyclers DNA size markers 1 kb DNA ladder Gibco BRL 15615 016 1 1 Agarose EtBr gel containing 0 1 ug ml ethidium bromide Proteinase K digestion Sterile 0 5 ml microcentrifuge tubes 95 ethanol e 80 ethanol cDNA size fractionation e 1 5 ml sterile microcentrifuge tubes Ring stand with small clamp for holding column e 1 1 agarose EtBr gel 0 1 ug ml ethidium bromide e 95 ethanol 20 1 xylene cyanol phage packaging phage packaging extract Several are commercially available Choose a packaging system that will give you at least 1 x 10 pfu ug of DNA PCR Insert Screening optional e A TriplEx LD Insert Screening Amplimer Sets Routine plating and culture of E coli e Kanamycin stock solution 25 mg ml in H20 500X Store at 20 C e Tetracycline stock solution 15 mg ml in H50 1000X Clontech Laboratories Inc www clontech com Protocol No PT3000 1 12 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Ill Additional Materials Required continued Store at 20 C Chloramphenicol stock solution 34 mg ml in 10096 ethanol 1000X
32. d stably in E coli Do not attempt blue white screening with strain BM25 8 Conversion Protocol Pick a single isolated colony from the working stock plate of BM25 8 host cells Step VII B 4 and use it to inoculate 10 ml of LB broth in a 50 ml test tube or Erlenmeyer flask Incubate at 31 C overnight with shaking at 150 rpm until the ODgoo of the culture reaches 1 1 1 4 2 Add 100 ul of 1 M MgCl to the 10 ml overnight culture of BM25 8 10 mM final concentration of MgCl 3 Pick a well isolated positive plaque from secondary or tertiary screening plates and place it in 350 ul of 1X lambda dilution buffer Vortex the plaque and incubate at 37 C for 3 4 hr without shaking 200 250 rpm Alternatively allow phage to elute at 4 C overnight 4 In a 20 ml test tube combine 200 ul of overnight cell culture with 150 ul ofthe eluted positive plaque Reserve the remainder of the eluted plaque in case you need to repeat the conversion Incubate at 31 C for 30 min without shaking Add 400 yl of LB broth Incubate at 31 C for an additional 1 hr with shaking 225 rpm Using a sterile glass spreader spread 1 10 ul of infected cell sus pension on an LB carbenicillin plate to obtain isolated colonies and grow at 31 C Note Ampicillin can be used instead of carbenicillin but may result in more satel lite colonies 9 Pick several well isolated colonies from each clone and prepare plasmid DNA separately from each one
33. e cDNA to vector Sections VII A Package library in phage amp plate out see manufacturer s recommendations Titer unamplified library amp check recombinant clones Sections VII C amp D Amplify and titer library Sections VILE amp F Figure 4 Guide to using the SMART cDNA Library Construction Kit protocols Be sure to choose the appropriate protocol for your application Refer to Figure 5 and Section VIII for troubleshooting tips ONE TWO DAYS THIRD DAY Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 7 SMART cDNA Library Construction Kit User Manual V SMART cDNA Synthesis by LD PCR PLEASE READ ENTIRE PROTOCOL BEFORE STARTING Important This protocol is designed for library construction of SMART cDNA libraries using a PCR based protocol The minimum amount of starting material for cDNA synthesis is 50 ng of total RNA or 25 ng of poly At RNA In general the more RNA you start with the fewer PCR cycles will be required for the second strand synthesis see Table III Fewer thermal cycles are less likely to generate nonspecific PCR products and therefore are best for optimal cDNA and library quality Thus if your RNA sample is not limiting use the higher starting amounts of RNA shown in the table To construct SMART cDNA libraries from 1 ug of poly A RNA using primer extension consult the protocol in Section VI We strongly recommend that you perform a posit
34. ed lysate to yield 6 7 x 10 plaques per 150 mm plate Incubate in a 37 C water bath for 15 min Add 4 5 ml of melted LB MgSO soft top agar to each tube Quickly mix and pourthe bacteria phage mixture onto LB MgSO agar plates Swirl the plate quickly while pouring to promote even distribution of the melted agar over the plate Cool the plates at room temperature for 10 min to allow the top agar to harden Invert the plates and incubate at 37 C for 6 18 hr or until the plaques become confluent i e are touching each other Add 12 ml of 1X lambda dilution buffer to each plate Store plates at 4 C overnight The plaques are now ready to be pooled in 1X lambda dilution buffer to form an amplified library lysate On a platform shaker 50 rpm incubate the plates at room tem perature for 1 hr Pour the phage lysates into a sterile beaker This is the pooled amplified library lysate To clear the phage lysate of cell debris and to lyse any remaining intact cells a Mix the phage lysate well and pour it into a sterile 50 ml poly propylene screw cap tube b Add 10 ml of chloroform to the lysate c Screw on the cap and vortex for 2 min Clontech Laboratories Inc www clontech com Protocol No PT3000 1 34 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Vil SMART cDNA Library Protocols continued d Centrifuge in a Beckman J2 21 centrifuge at 7 000 rpm 5000 x g for
35. ental RNA may differ from those shown for the control reaction 3 Some low molecular weight material The ds cDNA generated using primer extension will contain some degraded RNA fragments 0 1 kb a strong signal can be seen at position of about 100 bp in Figure 6B These fragments are removed by the size fractionation columns see Section VI E amp Section VIII G Clontech Laboratories Inc www clontech com Protocol No PT3000 1 44 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Appendix B Converting ATriplEx2 to pTriplEx2 A Background The conversion of a ATriplEx2 clone to a pTriplEx2 plasmid involves in vivo excision and circularization of a complete plasmid from the recombinant phage The plasmid is released as a result of Cre recom binase mediated site specific recombination at the oxP sites flanking the embedded plasmid Figure 7 Release of the plasmid occurs auto matically when the recombinant phage is transduced into a bacterial host in which Cre recombinase is being expressed In this system E coli BM25 8 growing at 31 C providesthe necessary Cre recombinase activity Conversion may be performed on individual positive plaques picked from the secondary or tertiary screening plates The released version of the plasmid differs from pTriplEx2 by a 100 bp loxP insert at the Cla I site please refer to the Vector Information packet for a vector map and mcs The excised plasmid is propagate
36. entle inversion for 1 2 min Centrifuge at 14 000 rpm for 5 min to separate the phases Move the top aqueous layer to a clean 0 5 ml tube Discard the interface and lower layers Add 100 ul of chloroform isoamyl alcohol to the aqueous layer Mix by continuous gentle inversion for 1 2 min Centrifuge at 14 000 rpm for 5 min to separate the phases Clontech Laboratories Inc www clontech com Protocol No PT3000 1 26 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VI SMART cDNA Synthesis by Primer Extension continued 10 Move the top aqueous layer to a clean 0 5 ml tube Discard the interface and lower layers Add 10 ul of 3 M Sodium Acetate 1 3 ul of Glycogen 20 ug l and 260 ul of room temperature 9596 ethanol Immediately centrifuge at 14 000 rpm for 20 min at room temperature Note Do not chill the tube at 20 C or on ice before centrifuging Chilling the sample will result in coprecipitation of impurities 12 Carefully remove the supernatant with a pipette Do not disturb the pellet 13 Wash pellet with 100 ul of 80 ethanol 14 Air dry the pellet 10 min to evaporate off residual ethanol 15 Add 79 ul of deionized H O to resuspend the pellet D Sfil digestion 1 Combine the following components in a fresh 0 5 ml tube 79 ul cDNA Section VI C 15 above 10 ul 10X Sfi Buffer 10 ul Sfil Enzyme 1 ul 100X BSA 100 ul Total volume 2 Mix well Incubate the tube at 50 C f
37. esis we recommend that you estimate the integrity of the RNA by examining a sample on a denaturing formaldehyde agarose gel Total RNA from mammalian sources should appear as two distinct bands 28S and 18S ribosomal RNA at approximately 4 5 and 1 9 kb The ratio of intensities of the 28S and 18S rRNA bands should be 1 5 2 5 1 Intact mammalian poly At RNA should appear as a smear usually 0 5 12 kb with faint 28S and 18S rRNA bands The size distribution may be considerably smaller 0 5 3 kb for nonmammalian species e g plants insects yeast amphibians etc If your experimental poly A RNA appears significantly smaller than expected e g no larger than 1 5 kb we suggest you pre pare fresh RNA after checking your RNA purification reagents for RNase and other impurities If problems persist you may need to find another source of tissue cells Alternatively you may wish to use one of Clontech s Poly A RNAs Clontech Laboratories Inc www clontech com Protocol No PT3000 1 16 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual V SMART cDNA Synthesis Protocols 50 ng of total RNA or 25 ng of poly A RNA 1 pg poly A RNA researcher provides researcher provides Synthesize first strand cDNA Synthesize first strand cDNA Section V A Section VILA cDNA Synthesis by LD PCR cDNA by Primer Extension Section V B Section VI B Electrophorese a sample on a gel Sfi I digestion cDNA size fractionation Ligat
38. ess Before proceeding to the next step allow the sample to be fully absorbed into the surface of the matrix i e there should be no liquid remaining above the surface With 100 ul of column buffer wash the tube that contained the cDNA and gently apply this material to the surface of the matrix Allow the buffer to drain out of the column until there is no liquid left above the resin When the dripping has ceased proceed to the Clontech Laboratories Inc www clontech com Protocol No PT3000 1 22 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual V SMART cDNA Synthesis by LD PCR continued next step At this point the dye layer should be several mm into the column 8 Place the rack containing the collection tubes Section V E 1 un der the column so that the first tube is directly under the column outlet 9 Add 600 ul of column buffer and immediately begin collecting single drop fractions approximately 35 ul per tube in tubes 1 16 Cap each tube after each fraction is collected Recap the column after fraction 16 has been collected 10 Check the profile of the fractions before proceeding with the ex periment On a 1 1 agarose EtBr gel electrophorese 3 ul of each fraction separately in adjacent wells alongside 0 1 ug of a 1 kb DNA size marker Run the gel at 150 V for 10 min Running the gel longer will make it difficult to see the cDNA bands Determine the peak fractions by vi
39. ete analyze a 5 ul sample of the product on a 1 1 agarose EtBr gel alongside a 1 kb DNA size marker 0 1 ug The ds cDNA should appear as a 0 1 9 kb smear on the gel with some distinct bands corresponding to the abundant mRNAs for that tissue or cell source CDNA prepared from some tissues may not have distinct distinct bands especially if the mRNA is highly complex If your product does not appear as expected refer to the Troubleshooting Guide SectionVIII A B F Typical results obtained with Human Placenta Poly At RNA are shown in Appendix A 7 Proceed to the next step or store ds cDNA at 20 C until use C Proteinase K digestion oR WD Oo In a sterile 0 5 ml tube pipet 50 ul of amplified ds cDNA 2 3 ug making sure you pipet below the top oil layer of the PCR tube and add 2 ul of proteinase K 20 ug ul Store the remaining ds cDNA at 20 C up to 3 months Note Proteinase K treatment is necessary to inactivate the DNA polymerase activ ity This system was optimized for 2 3 ug 50 ul vol of ds cDNA for subsequent cloning and library construction procedures Too much ds cDNA gt 3 4 ug will yield a low titer library Refer to theTroubleshooting Guide Section VIII H for more information Mix contents and spin the tube briefly Incubate at 45 C for 20 min Spin the tube briefly Add 50 ul of Deionized H O to the tube Add 100 ul of phenol chloroform isoamyl alcohol and mix by con tinuous g
40. ethyformamide DMF Store at 20 C Long term library storage e 100 Dimethylsulfoxide DMSO e 100 Glycerol Clontech Laboratories Inc www clontech com Protocol No PT3000 1 14 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual IV General Considerations A Good Laboratory Practices 1 Wear gloves throughout the procedure to protect your RNA and cDNA samples from degradation by nucleases 2 When resuspending pellets or mixing reactions gently pipet the solution up and down or tap the bottom of the tube Spin tube briefly to bring contents to the bottom of the tube Do not vortex samples when resuspending pellets vortexing may cause shearing of your cDNA 3 Perform all reactions on ice unless otherwise indicated 4 Add enzymes to reaction mixtures last Make sure that the enzyme is thoroughly blended into the reaction mixture by gently pipetting the mixture up and down 5 Do not increase the size volume of any of the reactions All com ponents have been optimized for the volumes specified 6 Ethidium bromide is a carcinogen Use appropriate precautions in handling and disposing this reagent For more information see Sambrook et al 1989 Several EtBr disposable cartridges are also available such as Clontech s BondEx Ethidium Bromide Detoxifica tion Cartridge see Section X Related Products 7 Phenol is a corrosive Always wear gloves and protective clothing when handling solutions
41. gations combined you should obtain 1 2 x 10 independentclones The unamplified libraries can be stored at 4 C for 2 weeks Optional Ifyou obtained 1 2 x 10 clones you may wish to perform another ligation with the remaining cDNA Repeatthe ligation using the ratio of cDNA to vector of the initial three ligations that gave the best results Scale up the volumes of all reagents according to the amount of cDNA used Then package and titer this scaled up ligation If the titer is still low see Section VIII H for troubleshooting tips To increase the stability of your library first combine the packaging reactions from Section VII A 5 you should have gt 1 x 109 indepen dent clones then amplify the library as described in Section VII E The amplified library can be stored at 4 C for 6 7 months or at 70 C in 7 DMSO for at least one year B Bacterial Culture Plating E coli XL1 Blue and BM25 8 are provided as a stock in LB medium with 25 glycerol and can be stored indefinitely at 70 C 1 To recover the frozen cells streak a small portion 5 ul of the frozen stock onto an LB agar plate containing the appropriate antibiotic This is the primary streak plate Use LB tet for XL1 Blue stock plates e Use LB kan cam for BM25 8 stock plates Note When streaking bacterial cultures for use as stock plates use LB agar medium without MgSO Incubate at 37 C overnight Wrap plate in Parafilm and store at 4 C for
42. he banding pattern of your PCR producttothe 1 kb DNA size marker 0 1 ug run on the same gel For cDNA made from all mammalian RNA sources the overall signal intensity relative to the marker DNA should be roughly similar to that shown for the control ex perimentin Figure 6A If the intensity of the cDNA smearis much stronger than that shown for the control relative to 0 1 ug of size markers es pecially if no bands are distinguishable this may indicate that too many thermal cycles were used i e PCR overcycling has occurred see Section VIII D If the smear is much fainter and the size distribution is less than 4 kb then too few thermal cycles i e PCR undercycling may be the problem see Section VIII C Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 43 SMART cDNA Library Construction Kit User Manual Appendix A Typical Results continued 2 Several distinct bands corresponding to abundant transcripts The pattern of distinct bands shown in Figure 6A is characteristic of the PCR product made using the control human placenta poly A RNA A very strong smear of cDNA in the control reaction without the charac teristic distinct bands may beindicative of PCR overcycling see Section VIII D If the characteristic bands are present but weak this may be indicative of PCR undercycling see Section VIII C The number and position of the bands you obtain with your experimental RNA may d
43. ide The resulting full length ss cDNA contains the complete 5 end of the mRNA as well as the sequence complementary to the SMART IV Oligo which then serves as auniversal priming site SMART anchor in the subsequent amplification by LD PCR Chenchik et al 1998 Only those ss cDNAs having a SMART anchor sequence at the 5 end can serve as a template and can be exponentially amplified Incomplete cDNAs and cDNA transcribed from polyA RNA will lack the SMART anchor and will not be amplified Thus contamination by genomic DNA and poly A RNA is eliminated This selective amplification allows you to construct a cDNA library using nanogram amounts of either total RNA or poly A RNA with a high percentage of full length clones cDNA synthesis by Primer Extension In the second protocol Section VI cDNA synthesis employs primer exten sion Sambrook et al 1989 which replaces the LD PCR step in ds cDNA synthesis Figure 1 Similar to the first strand cDNA synthesis described above the CDS III 3 Primer is used to prime the first strand reaction and the SMART IV Oligo serves as a short extended template at the 5 end of the mRNA After first strand cDNA synthesis the primer extension step gener ates full length ds cDNA This protocol is ideal for researchers who are not limited by their starting material i e 1 ug or more poly A RNA Optimized reagents and conditions The SMART kit incorporates carefully optimized reagents and condi
44. ide a rough es timate of your target cDNA concentration On a 1 196 agarose gel you can compare the intensity of your ds cDNA with the cDNA synthesized from the control RNA provided with this kit Typically the control RNA generates 4 6 ug of ds cDNA by this protocol I Low lt 80 recombination efficiency The Sfil A amp B digested XTriplEx2 arms provided in the SMART Kit have been dephosphorylated background due to re ligation of nonre combinant vector molecules should be minimal This can be checked by performing a control ligation using vector alone no cDNA in Step VII A 1 A high titer combined with an apparently low recombination efficiency may indicate that your cDNA population contains a lot of small DNA fragments which are preferentially ligated into the vector If you suspect this may be the case repeat the cDNA synthesis and check the size distribution of the fractionated cDNA Also see Section VIII G J Small insert sizes If more than 5096 of your clones appear to have small insert sizes i e 0 4 kb itis likely that your cDNA preparation was not success fully size fractionated Small cDNA fragments unincorporated primers and primer dimers are preferentially ligated to the vector and should be completely removed before the vector ligation step Because this problem cannot be retroactively solved you will have to repeat the cDNA synthesis procedure However before you do so read SectionVIll G above
45. iffer from those shown for the control reaction Furthermore cDNA prepared from some mammalian tissue sources e g human brain spleen and thymus may not display distinct bands due to the very high complexity of the poly A RNA 3 Some low molecular weight material Most raw PCR reaction products will contain some small cDNA fragments 0 4 kb and some very small i e 0 1 kb non cDNA contaminants as seen in Figure 6A The non cDNA contaminants include unincorpo rated primers and SMART oligonucleotides as well as primer dimers A preponderance of material in the lower part of the gel i e 0 1 kb may indicate that PCR overcycling has occurred see Section VIII D B Results for Primer Extension Figure 6B 1 A moderately strong smear of cDNA from 0 1 to 9 kb or more Comparetheintensity ofthebanding pattern ofyourdscDNAproducttothe 1 kb DNA size marker 0 1 ug run on the same gel Primer extension generates a longer smear than the ds cDNA synthesized using LD PCR If your cDNA is less than 3 kb itis possible that your RNA is degraded see Section VIII F 2 Weak bands correspond to abundant transcripts Primer extension requires more poly A RNA for first strand synthesis than required by LD PCR therefore the bands in Figure 6B appear weaker than the bands in Figure 6A Weaker bands correspond to a higher complexity of the poly A starting material The number and position of the bands you obtain with your experim
46. ive control cDNA synthe sis with the human placenta poly A RNA provided in parallel with your experimental cDNA synthesis Performing a control at least once will verify that all the components are working properly This is especially important because it may not be possible to visualize your first strand reaction prod uct on a gel Furthermore performing the control reaction through the PCR step in parallel with your experiment will allow you to evaluate the yield and size distribution of the ds cDNA synthesized from your RNA sample Performing the control through the library construction steps will also help you troubleshoot any problems as they arise Notes f necessary the procedure can be stopped after first strand synthe sis by placing the reaction mixture at 20 C and also at any ethanol precipitation step after the precipitated DNA has been centrifuged or when the supernatant has been removed The dry or resuspended DNA pellet can be stored at 20 C for up to 3 months Forthefirst strand synthesisand the PCR all components and reaction vessels should be pre chilled on ice A First Strand cDNA Synthesis 1 Combine the following reagents in a sterile 0 5 ml microcentrifuge tube 1 3 ul RNA sample 0 025 0 5 ug poly At or 0 05 1 0 ug total RNA Forthecontrolreaction use1ul 1uglofthecontrol RNA 1 ul SMART IV Oligonucleotide 1 ul CDS IIl 3 PCR Primer If total volume is lt 5 ul add deionized H O to bring volume up to 5 y
47. l Clontech Laboratories Inc www clontech com Protocol No PT3000 1 18 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual V SMART cDNA Synthesis by LD PCR continued Mix contents and spin the tube briefly in a microcentrifuge Incubate the tube at 72 C for 2 min Cool the tube on ice for 2 min Spin the tube briefly to collect the contents at the bottom Add the following to each reaction tube 2 0 ul 5X First Strand Buffer 1 0 ul DTT 20 mM 1 0 ul dNTP Mix 10 mM 1 0 ul MMLV ReverseTranscriptase 10 0 ul Total volume Please see Addendum PT3980 4 for details on the choice of RT enzyme 7 Mix the contents of the tube by gently pipetting and briefly spin ning the tube 8 Incubate the tube at 42 C for 1 hr in an air incubator or a hot lid thermal cycler Note If you use a water bath or thermal cycler for this incubation cover the reaction mixture with one drop of mineral oil before you close the tube This will prevent loss of volume due to evaporation 9 Place the tube on ice to terminate first strand synthesis 10 If you plan to proceed directly to the PCR step take a 2 ul aliquot from the first strand synthesis and place it in a clean prechilled 0 5 ml tube Place the tube on ice and proceed to Step V B If you used mineral oil in your first strand reaction tube be careful to take the 2 ul sample from the bottom of the tube to avoid the oil Any first strand reaction mixture th
48. l provides a more traditional protocol for researchers with abundant amounts of starting material i e 1 ug or more poly A RNA Both protocols utilize the patented SMART IV Switching Mechanism At 5 end of RNA Transcript Oligonucleotide in the first strand synthesis to generate high yields of full length double stranded ds cDNA Figure 1 Full length cDNA with complete 5 ends All commonly used cDNA synthesis methods rely on the ability of reverse transcriptase RT to transcribe mRNA into single stranded ss DNA in the first strand reaction In some cases RT terminates before transcribing the complete mRNA sequence Thisis particularly trueforlong mRNAs especially if the first strand synthesis is primed with oligo dT primers only or if the mRNA contains abundant secondary structures In addition conventional cDNA cloning procedures use the T4 DNA polymerase to generate blunt cDNA ends after second strand synthesis As a result under represented 5 ends of genes in cDNA populations tend to be 5 30 nucleotides shorter than the original mRNA D Allessio 1988 The SMART protocols are designed to preferentially enrich for full length cDNAs while eliminating T4 DNA polymerase and adaptor ligation SMART libraries are proven to contain a higher percentage of full length clones than libraries constructed by con ventional methods October 1998 Clontechniques or other full length cDNA synthesis protocols Okayama amp Berg 1982 Kat
49. n Kit User Manual VII SMART cDNA Library Protocols continued TABLE V PLATING DILUTIONS FOR TITERING AN AMPLIFIED LIBRARY 1X Lambda 1X Lambda Bacterial Phage Tube Dilution Buffer Overnight Culture Dilution 2 1 100 ul 200 ul 5 yl 2 100 ul 200 ul 10 ul 3 100 ul 200 ul 20 ul 4 Control 100 ul 200 ul 0 ul 5 Incubate tubes in a 37 C water bath for 15 min 6 11 Add 3 ml of melted 45 C LB MgSO top agar to each of the four tubes Quickly mix and pour the contents from each tube onto separate LB MgSO agar plates Swirl the plates quickly after pouring to promote an even distribution of the melted agar Cool plates at room temperature for 10 min to allow the soft agar to harden Incubate plates inverted position at 37 C for at least 6 7 hr 10 Count the plaques and calculate the titer pfu ml as follows pfu ml number of plaques x dilution factor x 10 ul ml ul of diluted phage plated n this case the dilution factor 1 x 104 A successfully amplified library will have a very high titer 1070 pfu ml Clontech Laboratories Inc www clontech com Protocol No PT3000 1 36 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VIII Troubleshooting Guide Obtain or isolate total or poly A RNA Section IV B Recommended Check quality Repeat Section IV B of RNA on a gel Section IV B if necessary Repeat Sections IV B Synthesize first strand cDNA Tro
50. nmatchable your RNA starting material may be degraded very impure or too dilute Check the quality and quantity of your RNA by running a sample on a gel as recommended in Sec tion IV B 3 If the RNA seems too dilute but otherwise of good quality restart the experiment using more RNA If the RNA seems degraded restart the experiment using a fresh lot or preparation of RNA Also check the stability of your RNA by incubating a small sample at 37 C for 2 hr Run it on a gel parallel to a fresh unincubated sample If the RNA appears to be unstable it will yield poor results If this is the case reisolate the RNA using a different method see Section X for Related Products Please note that problems with your RNA are eas ily diagnosed if you perform parallel reactions using the control RNA provided in this kit C Low yield of ds cDNA product using LD PCR Step V B 6 1 Toofew thermal cycles used in the PCR step i e PCRundercycling Another indication of PCR undercycling is a cDNA size distribution lt 4kb ifthe mRNA source was mammalian For some sources such as many insect species the normal mRNA size distribution may be 2 3 kb If you suspect undercycling incubate the PCR reaction mixture for two more cycles and recheck the product If you already used the maximum recommended number of cycles indicated in Table III increase by three more cycles If increasing the number of cycles does not improve the yield of PCR product
51. o et al 1994 Thus clones isolated from SMART cDNA libraries contain sequences corresponding to the complete 5 untranslated region of the mRNA ibid SMART preserves the complete 5 mRNA sequence Libraries made with SMART cDNA can facilitate preliminary mapping of transcription start sites FFomont Racine 1993 due to the high percentage of full length clones However SMART cDNA libraries may not be suitable for immunoscreening for certain proteins In some cases 5 untranslated regions UTRs may contain stop codons in frame with the initiating transla tion start site in the expression vector Clontech Laboratories Inc www clontech com Protocol No PT3000 1 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Introduction continued Poly A RNA B AVAAAUUUVUUV polyA 3 GG 5 SMART IV Oligonucleotide SfilA c66 ga cce 5 AVAVAVAAVAVUAUMV polyA CDS III Primer SfilB First strand synthesis coupled with dC tailing by RT Y Incomplete transcripts or premature termination of RT 5 AVAAVAUAUUUS polyA SMART Template switching Ys cDNA Synthesis amp extension by RT v Truncated cDNA 5mm GGG PAPAS polyA Lacks 5 primer binding site CC N Cannot be converted LD PCR primer extension to ds DNA or cloned from 50 ng of total RNA from 1 g of poly A RNA e Enriched full length ds cDNA Sfil digestion Y m SMART SfilA SfilB
52. o evaporation 9 Place the tube on ice to terminate first strand synthesis 10 Add 1 ul of Sodium Hydroxide to the tube 11 Incubate the tube at 68 C for 30 min 12 Place the tube on ice and proceed to Step VI B Any first strand reaction mixture that is not used right away should be placed at 20 C First strand cDNA can be stored at 20 C for up to three months B ds cDNA synthesis by primer extension 1 Preheat the PCR thermal cycler to 95 C 2 Combine the following components 11 ul First Strand cDNA from Step VIA 12 71 ul Deionized H O 10 ul 10X Advantage 2 PCR Buffer 2 ul 50X dNTP Mix 2 ul 5 PCR Primer 2 ul CDS III 3 PCR Primer 2 ul 50X Advantage 2 Polymerase Mix 100 ul Total volume 3 Mix contents by gently flicking the tube Centrifuge briefly to col lect the contents at the bottom of the tube 4 Overlay the reaction mixture with 2 drops of mineral oil if necessary Cap the tube and place it in a preheated 95 C thermal cycler Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 25 SMART cDNA Library Construction Kit User Manual VI SMART cDNA Synthesis by Primer Extension continued 5 6 Commence primer extension using one of the following pro grams GeneAmp 480 GeneAmp 2400 9600 e 72 C 10 min e 72 C 10 min e 95 C 1 min e 95 C 20 sec e 3 cycles e 3 cycles 95 C 15sec 95 C 5sec 68 C 8 min 68 C 38min When the reaction is compl
53. ombinant Clones In XATriplEx2 as in all other X expression vectors the cloning site is embedded in the coding sequence for the a polypeptide of f galacto sidase lacZ This makes it possible to use lacZ a complementation Sambrook et al 1989 to easily identify insert containing phage by transducing an appropriate host strain such as E coli XL1 Blue and screening for blue plaques on medium containing IPTG and X gal Do not attempt blue white screening with strain BM25 8 To perform blue white screening in E coli XL1 Blue follow the proce dure for titering an unamplified library on LB MgSO plates Section VII C except add IPTG and X gal to the melted top agar before plating the phage bacteria mixtures For every 2 ml of melted top agar use 50 ul each of the IPTG and X gal stock solutions Section III Aim for 500 1 000 plaques 90 mm plate Incubate plates at 37 C for 6 18 hr or until plaques and blue color develop The ratio of white recombinant to blue nonrecombinant plaques will give you a quick estimate of recombination efficiency A successful ligation using cDNA synthesized from the Control Poly At RNA provided will resultin at least 80 recombinants If your recombination efficiency for the control is lower than this see Section VIII I for troubleshooting tips Optional PCR insert screening of SMART PCR cDNA Library To test for ligation efficiency we recommend screening your cDNA insert using Clontech s Advan
54. on of the Sfil A amp B recognition sequences Another feature of the SMART Kit is the incorporation of asymmetrical Sfil restriction enzyme sites A amp B Figure 2 at the 5 and 3 cDNA ends respectively Figure 1 After digestion with Sfil and size fractionation us ing CHROMA SPIN Columns SMART cDNA is ready for ligation into the Sfil digested ATriplIEx2 Vector ATriplEx2 which contains the asymmetrical Sfil sites A amp B in the MCS eliminates adaptor ligation and facilitates di rectional cloning Furthermore Sfil sites are extremely rare in mammalian DNA therefore all SMART cDNAs remain intact after Sfil digestion Methyla tion steps are eliminated valuable internal restriction sites are preserved Typical results of cDNA synthesis using the Human Placenta Poly At RNA provided are shown in Appendix A Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 7 SMART cDNA Library Construction Kit User Manual I Introduction continued cDNA insert IN vector E S UTRompa RBS ATG 4 RBS ATGjao7 dT T7 lacZ Y vector H A V Translation in all three reading frames 1 Normal translation f Met from ompA start codon N 2 Peptide resulting from slippage of RNA polymerase amp or ribosome 3 Normal translation from lacZ start codon Figure 3 Generation of polypeptides from all three reading frames in a single recombinant XTriplEx
55. oom tempera ture 15 Carefully remove the supernatant with a pipette Do not disturb the pellet 16 Briefly centrifuge the tube to bring all remaining liquid to the bot tom 17 Carefully remove all liquid and allow the pellet to air dry for 10 min 18 Resuspend the pellet in 7 ul of Deionized H O and mix gently The Sfil digested cDNA is now ready to be ligated to the Sfil digested dephosphorylated ATriplEx2 Vector provided Proceed to Section VII or store cDNA at 20 C until the ligation step Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 29 SMART cDNA Library Construction Kit User Manual Vil SMART cDNA Library Protocols A Ligation of cDNA to XTriplEx2 Vector The ratio of cDNA to vector in the ligation reaction is a critical factor in determining transformation efficiency and ultimately the number of independent clones in the library The optimal ratio of cDNA to vector in ligation reactions must be determined empirically for each vector cDNA combination To ensure that you obtain the best possible library from your cDNA set up three parallel ligations using three different ratios of cDNA to vector as shown in Table IV Important note Use a X phage packaging system that will give you at least 1 x 10 pfu ug of DNA Follow the supplier s recommended protocol and perform a parallel packaging reaction with the control insert provided in the packaging kit The following pro
56. or 2 hr 3 Add 2 ul of 1 xylene cyanol dye to the tube above Mix well E cDNA size fractionation by CHROMA SPIN 400 1 Label sixteen 1 5 ml tubes and arrange them in a rack in order 2 Prepare the CHROMA SPIN 400 column for drip procedure a Remove the CHROMA SPIN column from the refrigerator and warm at room temperature for about 1 hr Invert the column several times to completely resuspend the gel matrix b Remove air bubbles from the column Use a 1000 ul pipettor to resuspend the matrix gently avoid generating air bubbles Then remove the bottom cap and let the column drip naturally If the column does not drain after 3 min recap the top cap This pressure should cause the column to drain c Attach the column to a ring stand d Let the storage buffer drain through the column by gravity flow until you can see the surface of the gel beads in the column ma trix The top of the column matrix should be at the 1 0 ml mark on the wall of the column If your column contains significantly less matrix adjust the volume of the matrix to the 1 0 ml mark 1 Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 21 SMART cDNA Library Construction Kit User Manual VI SMART cDNA Synthesis by Primer Extension continued 10 using matrix from another column an extra column is provided for this purpose e The flow rate should be approximately 1 drop 40 60 sec The vol ume of 1 drop
57. p media additives 11 Table Ill Relationship between amount of RNA starting material amp optimal number of thermal cycles 20 Table IV Ligations using three different ratios of cDNA to vector 30 TableV Plating dilutions for titering an amplified library 36 List of Figures Figure 1 Flowchart of the SMART cDNA Library Construction Kit protocols Figure 2 Sequence of Sfil A amp B recognition sites Figure 3 Generation of polypeptides from all three reading frames in a single recombinant ATriplEx2 clone Figure 4 Guide to using the SMART cDNA Library Construction Kit protocols 17 Figure 5 Guide to troubleshooting SMART cDNA synthesis 37 Figure 6 Typical Results ds cDNA synthesized using the SMART control reagents amp protocols 43 Figure 7 Conversion of recombinant XTriplEx2 to the corresponding pTriplEx2 47 Figure 8 Restriction map of XATriplEx2 48 Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 3 SMART cDNA Library Construction Kit User Manual l Introduction The SMART cDNA Library Construction Kit provides a method for produc ing high quality full length cDNA libraries from nanograms of total or poly A RNA This kit contains two separate protocols allowing you to choose a method based on your starting material The first protocol employs a novel PCR based method for researchers limited by their starting material i e 50 ng of total RNA The second protoco
58. repeat the PCR de novo using a fresh 2 ul aliquot of the first strand product 2 If you still obtain a low yield of PCR product it may be due to a low yield of first strand cDNA Possible problems with the first strand reaction include a mistake in the procedure such as using a suboptimal incubation temperature or omitting a component or insufficient RNA in the reaction It is also possible that the RNA has been partially degraded by contaminating RNases before or during the first strand synthesis Reminder problems with the first strand cDNA synthesiscan be more easily diagnosed if you perform parallel reactions using the control RNA provided in the kit If good results were obtained with the control RNA but not with your experimental RNA then there may be a problem with your RNA See Section VIII Clontech Laboratories Inc www clontech com Protocol No PT3000 1 38 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VIII Troubleshooting Guide continued B above The easiest way to check the quality of the first strand cDNA is by using a small sample of it as a PCR template with 3 and 5 gene specific primers such as Human f Actin Control Amplimers Cat Nos 639001 amp 639002 If the first strand synthesis has been suc cessful a PCR product of the expected size will be generated D No distinct bands distinguishable in the ds cDNA product using LD PCR Section V B 6 As explained in Appendix
59. ry Construction Kit October1998 Clontechniques XIII 4 12 13 Note SMART technology was originally described as Capfinder Clontech Laboratories Inc www clontech com Protocol No PT3000 1 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Appendix A Typical Results of ds cDNA Synthesis A kb M ds cDNA B kb M ds cDNA DON WwW BOON i ON wo BATON es 0 5 Figure 6 Typical results ds cDNA synthesized using the SMART control reagents and protocol 1 ul 1 0 ug of the Control Human Placenta poly A RNA provided in the kit was used as start ing material in a first strand cDNA synthesis Panel A 2 ul of the ss cDNA served as template for LD PCR based second strand synthesis using 15 thermal cycles Panel B 10 ul of ss cDNA served as a template for primer extension based second strand synthesis using 3 cycles A 5 ul sample of the ds cDNA product was electrophoresed on a 1 1 agarose EtBr gel Lane M 1 kb DNA size marker 0 1 ug loaded The arrow indicates a strong band at 900 bp typically seen for human placenta poly A RNA Figure 6A amp 6B shows typical gel profiles of ds cDNA synthesized using the control human placenta poly A RNA and the SMART protocols Typical results for ds cDNA synthesis using LD PCR or Primer Extension should have the following characteristics A Results for LD PCR Figure 6A 1 A moderately strong smear of cDNA from 0 1 to 4 kb Comparethe intensity oft
60. sualizing the intensity of the bands under UV Collect the first three fractions containing cDNA in most cases the fourth fraction containing cDNA is usable Make sure the fourth fraction matches your desired size distribution Pool the above fractions in a clean 1 5 ml tube Add the following reagents to the tube with 3 4 pooled fractions containing the cDNA 105 140 ul respectively 1 10 vol Sodium Acetate 3 M pH 4 8 1 3 ul Glycogen 20 mg ml 2 5 vol 9596 ethanol 20 C 12 Mix by gently rocking the tube back and forth 13 Place the tube in 20 C or a dry ice ethanol bath for 1 hr Optional you may incubate at 20 C overnight which may result in better recovery 14 Centrifuge the tube at 14 000 rpm for 20 min at room tempera ture 15 Carefully remove the supernatant with a pipette Do not disturb the pellet 16 Briefly centrifuge the tube to bring all remaining liquid to the bot tom 17 Carefully remove all liquid and allow the pellet to air dry for 10 min 18 Resuspend the pellet in 7 ul of Deionized H O and mix gently The Sfil digested cDNA is now ready to be ligated to the Sfil digested dephosphorylated ATriplEx2 Vector provided Proceed to Section VII or store cDNA at 20 C until the ligation step 1 Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 23 SMART cDNA Library Construction Kit User Manual VI SMART cDNA Synthesis by Primer Extension
61. tage 2 PCR Kit and ATriplEx LD Insert Screening Amplimers Section X Related Products Efficient ligation of the cDNA to the ATriplEx2 Vector should result in more than 80 recombinants Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 33 SMART cDNA Library Construction Kit User Manual VII SMART cDNA Library Protocols continued E Library Amplification The number of plates required depends on how many independent clones are in the library to be amplified A general guideline is to aim for 6 7 x 10 clones or plaques per 150 mm plate when working with XTriplEx2 thus a library of 1 x 108 clones will require 20 plates If you are using another vector with a larger or smaller plaque size the optimal number of clones per plate may be different 10 1 1 Pick a single isolated colony from the primary working plate of XL1 Blue Section VII B 5 above and use it to inoculate 15 ml of LB MgSO maltose broth Incubate at 37 C overnight with shaking 140 rpm until the ODgo9 of the culture reaches 2 0 Centrifuge the cells for 5 min at 5 000 rpm pour off the supernatant and resus pend the pellet in 75 ml of 10 mM MgSO Plan the number of LB MgSO agar plates you will need Warm and dry them as explained in the note to Section VII C above Set up the required number of 4 ml tubes with 500 ul of overnight bacterial culture from Section VII E 1 above and enough dilut
62. ted top agar should be at 45 C higher temperatures will kill the bacteria Before plating with top agar the agar plates should be prewarmed to 37 C and the agar surface free of excess moisture droplets To dry the plates remove the lids and shake off excess droplets from the inside of the lids Just prior to use place the agar plates inverted and partially uncovered in a 37 C incubator to warm Freshly pre pared plates at room temperature will be warmed to 37 C in 10 15 min plates that have been stored at 4 C will require about 1 hr to warm up Do not over dry the plates When preparing media for phage transductions or plaque titering use recipes contain ing 10 mM MgSO for optimal adsorption of phage to bacteria For the same reason add 0 296 maltose to the LB broth when growing overnight bacterial cultures for transduction titering 1 Pick a single isolated colony from the working stock plate Section VII B 5 and use it to inoculate 15 ml of LB MgSO maltose broth ina 50 ml test tube or Erlenmeyer flask Incubate at 37 C overnight while shaking at 140 rpm until the OD o of the culture reaches 2 0 Centrifuge the cells at5 000 rpm for 5 min Pour off the supernatant and resuspend the pellet in 7 5 ml of 10 mM MgSO 2 Plan the number of 90 mm LB MgSO plates you will need Warm and dry them as explained in the note above 3 Make appropriate dilutions of each of the packaging extracts from SectionVII A 4 in
63. tions that improve the efficiency of all library construction steps A specially formulated first strand buffer and modified lock docking oligo dT primers Borson etal 1992 Chenchik etal 1994 optimize the efficiency of first strand synthesis Lock docking primers are designed to anneal to the junction of the poly A tail and the encoded transcript This increases the efficiency of cDNA synthesis by eliminating unnecessary reverse transcription of long stretches of poly A Clontech Laboratories Inc www clontech com Protocol No PT3000 1 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual Introduction continued We strongly recommend the use of the Advantage 2 PCR Kits Cat Nos 639206 amp 639207 for PCR amplification The specially formulated Advan tage 2 Polymerase Mix is optimized for efficient accurate and convenient amplification of cDNA templates by LD PCR The mix contains TITANIUM Taq DNA Polymerase a nuclease deficient N terminal deletion of Tag DNA polymerase plus TagStart Antibody to provide automatic hot start PCR Kellogg et al 1994 and a minor amount of a proofreading polymerase This polymerase system allows you to efficiently amplify full length cD NAs with a fidelity rate significantly higher than that of conventional PCR Barnes 1994 SfilA site SfilB site 5 GGCCATTACGGCC 3 5 GGCCGCCTCGGCC 3 3 CCGGTAATGCCGG 5 3 CCGGCGGAGCCGG 5 Figure 2 Comparis
64. tocol is optimized for ligation of SMART cDNA to the ATriplEx2 DNA provided 1 Set up a test ligation to determine the efficiency of ligating the vec tor to the Control Insert Use 1 ul of vector 1 ul of Control Insert 1 5 ulof Deionized H5O and other reagents listed inTable IV Incubate the test ligation at 16 C overnight Perform a X phage packaging reaction and titer the resulting phage Section VII C You should obtain 21 x 107 pfu ug of input vector 2 Label three 0 5 ml tubes and add the indicated reagents Table IV Mix the reagents gently avoid producing air bubbles Spin tubes briefly to bring contents to the bottom of the tube TABLE IV LIGATIONS USING THREE DIFFERENT RATIOS OF cDNA TO PHAGE VECTOR Component 1st ligation pl 2nd ligation pl 3rd ligation pl cDNA 0 5 1 0 1 5 Vector 500 ng ul 1 0 1 0 1 0 10X Ligation Buffer 0 5 0 5 0 5 ATP 10 mM 0 5 0 5 0 5 T4 DNA Ligase 0 5 0 5 0 5 Deionized H O 2 0 1 5 1 0 Total volume ul 5 0 5 0 5 0 To set up a test ligation use the volumes indicated in Section VII A 1 above Clontech Laboratories Inc www clontech com Protocol No PT3000 1 30 Version No PR7Y2399 SMART cDNA Library Construction Kit User Manual VII SMART cDNA Library Protocols continued Incubate tubes at 16 C overnight Perform a separate A phage packaging reaction for each of the ligations Titer each of the resulting libraries Section VII C From the three li
65. ubleshooting VA or VLA if necessary Section V A amp VI A see Section VIII A amp B check stability of RNA ds cDNA synthesis by LD PCR or by Primer Extonsian If undercycled incubate for 3 5 Repeat Sections IV B V A more cycles and recheck product or VB if necessary check stability of RNA i If overcycled repeat Section V B Check sp of ds x with a fresh aliquot of first strand Product ona ge DNA using fewer cycles Proceed to next step if results normal i For Primer extension troubleshooting see Section VIII F Check titer and recombinants of Troubleshooting library Sections VII C amp D see Sections VIII H amp I Figure 5 Guide to troubleshooting SMART cDNA synthesis Please refer to the text for further information on troubleshooting procedures Protocol No PT3000 1 www clontech com Clontech Laboratories Inc Version No PR7Y2399 7 SMART cDNA Library Construction Kit User Manual VIII Troubleshooting Guide continued A No ds cDNA product One or more essential reagents may have been inadvertently omitted from the first strand synthesis or the ds cDNA synthesis Repeat both of these steps being careful to check off every item as you add it to the reaction B Size distribution of ds cDNA product is less than expected ds cDNA size distribution should be similar to your mRNA which typi cally appears within the range of 0 5 10 kb on an agarose EtBr gel If the size distributions are u
66. ure commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc SMART Technology is covered by U S Patent Nos 5 962 271 and 5 962 272 For Profit and Not For Profit purchasers of SMART Products are entitled to use the reagents for internal research However the following uses are expressly prohibited 1 performing services for third parties 2 identifying nucleic acid sequences to be included on nucleic acid arrays blots or in libraries or other cDNA collections which are then sold to third parties Reproduc tion modification reformulation or resale of the reagents provided in SMART Products is not permitted For information on licensing SMART Technology for commercial purposes please contact a licensing representative by phone at 650 919 7320 or by e mail at licensing clontech com Use of the Sfil cloning strategy is licensed under U S Patent No 5 595 895 U S Patent No 5 436 149 for LATechnology is owned by TAKARA BIO INC Milli O is a trademark of Millipore NucleoTrap and NucleoSpin are registered trademarks of MACHEREY NAGEL GmbH and Co KG Parafilm is a registered trademark of American National Can Company GeneAmp is a registered trademark of Applera Corporation or its subsidiaries in the US and or certain other countries Clontech the Clontech logo and all other trademarks are the property of Clontech Laboratories Inc unless noted otherwise

Download Pdf Manuals

image

Related Search

Related Contents

User Manual AIR30 final  T'nB Guppy 3 VIP UK  GPS AutoSteer System Installation Manual - Terre-net  

Copyright © All rights reserved.
DMCA: DMCA_mwitty#outlook.com.