Director™ Universal Cloning System - Sigma
Contents
1. Using 1 ul 50 ng of vector mix insert vector at a molar ratio of 1 1 to 10 1 Transformation of ligation product Add 1 2 ul of ligation product to one tube of competent cells according to the manufacturers instructions If using the recommended GC5 Competent Cells Product Code G3169 place the tube on ice for 30 minutes Heat the tube for 45 seconds in a 42 C water bath Place the tube on ice for 2 minutes Add 450 ul of room temperature SOC medium to the tube Shake the tube at 37 C 225 rpm for 1 hour Plate 100 ul on a LB Amp plate and grow at 37 C overnight PCR verification of transformants A variety of methods can be used to verify positive transformants including restriction digestion sequencing and PCR Section 11 3 contains a PCR based rapid verification procedure suitable for screening a large number of transformants using verification primers provided in the Director Ready Vector Kits Version Date 10 05 05 11 gt W iL A m S gt m D Y A le O m E A m le A m X U m E m z o m D E o m A N Director Universal Cloning System Manual 9 0 DIRECTOR READY VECTOR SETS 9 1 Reagents Provided The Director Ready pFLAG MAC Set is used for cloning and cytoplasmic expression of N terminal Met FLAG fusion proteins in E coli Reagents provided in the kit are sufficient for 20 reactions Kit Component Product Number Quantity Director Ready p2. 14 11 3 Verification of Transformants eren enne trennen nennen rr nennen 14 MA elle 14 11 9 2 A e EC 14 11 3 3 POR profile 5 oit ce epe oi os etn iecore in pide LOG betae De tee b ea e EO EE cen 15 11 3 4 Gel electrophoresis reete nnne nemen sr nn inneren nnns 15 11 3 5 Expected results oett rtt REFERS ERG c DERE e ERI SER TERR uaa ERE REL ee 15 11 3 6 Verification of ligation reactions optional 16 12 0 Troubleshooting Guide rire drid Eun ERE eR TRA EE RR ENEE RARE ENER 17 13 0 References add Ce Fera Lade eu a DEE 19 Version Date 10 05 05 1 Director Universal Cloning System Manual 14 0 Related Products siria rx eei in e in 2O APPENDIX 1 Cleavage Sites PCR Primer Sequences for most Commonly Used 5 Overhang producing Restriction Enzymes eese nennen nnne 2 1 APPENDIX 2 pFLAG MAC Bacterial Expression Vector Information 22 APPENDIX 3 pFLAG CMV 2 Mammalian Expression Vector Information 23 APPENDIX 4 Verification Primer SEQuencess cccccecceeeeeseeeeeeeeeeeeeeeeneeeeeeeeseseseaneeeeeeeeseesseeneeseseeenees 24 LICENSE AGREEMENTS NOTICE TO PURCHASER LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 789 224 5 618 711 6 127 155 and claims outside the US correspond
3. Bama Sial Tee ATAlGAT CTG A TA TCG GTA CCA crc GAC Tic T AGA GGA TCC Tece GT 3 AGCiTAT CTA GAC TAIT AGC CAT GGT CAG C Tic AGA TCiT CCT AGIG IGCC CA 5 SER ILE ASP LEU ILE SER VAL PRO VAL ASP SER ARG GLY SER ARG Multiple Cloning Region Version Date 10 05 05 23 Director Universal Cloning System Manual APPENDIX 4 VERIFICATION PRIMER SEQUENCES Verification primer BF and BR BF 5 TGG CAA ATA TTC TGA AAT GAG C 3 BR 5 CTA CGG CGT TTC ACT TCT GAG T 3 The Verification Primer BF Product Code P5361 and BR Product Code P5486 are designed for double stranded or single stranded DNA sequencing of FLAG fusion construct of prLAG MAC expression vector The Verification Primer BF and BR may also be used for PCR mediated insert verification Verification Primer MF and MR MF 5 AAT GTC GTA ATA ACC CCG CCC CGT TGA CGC 3 MR 5 TAT TAG GAC AAG GCT GGT GGG CAC 3 The Verification Primer MF Product Code P2987 and MR Product Code P3112 are designed for double stranded or single stranded DNA sequencing of FLAG fusion construct of pFLAG CMV expression vectors The Verification Primer MF and MR may also be for PCR mediated insert verification 24 Version Date 10 05 05
4. containing H20 10 ul JumpStart REDTaq ReadyMix DNA polymerase 2X 0 4 ul Verification primer F 20 pmol l or 20 uM stock 0 4 ul Verification primer R 20 pmol ul or 20 uM stock 20 ul X number of reactions 14 Version Date 10 05 05 11 3 3 PCR profile Initial denaturation 30 cycles Denaturation Annealing Extension Final extension Soak 94 C 5 minutes 94 C 30 seconds 62 C 30 seconds 68 C 3 minutes 68 C 10 minutes 4 C 11 3 4 Gel electrophoresis Director Universal Cloning System Manual Load 5 ul PCR marker in lane 1 of a 0 8 1 agarose gel Load 10 ul of each PCR in the remaining lanes Run the gel in 1x TBE buffer until the tracking dye is 1 cm from the bottom of the gel 11 3 5 Expected results The Verification PCR primers provided with the kits flank the MCS regions of Director Ready vectors Therefore after PCR the negative transformants resulting from self ligation will produce a 0 4 kb product whereas the positive transformants should result in a product equal to 0 4 kb plus the size of insert e g a 1 8 kb product for the control insert If there is no visible PCR product the PCR was not successful Alternatively one of the verification PCR primers could be used in combination with an insert specific primer to check for the presence and orientation of the insert The drawback of this approach is that it cannot determine whether a negative result was caused by th
5. content of primers and complexity of the template DNA are important considerations in successful PCR We recommend that Tm of your primers be between 65 75 C with GC content greater than 4096 6 3 DNA Polymerases The kit uses JumpStart REDAccuTaq LA DNA polymerase for PCR amplification This special enzyme blend offers several advantages over conventional Taq polymerases excellent amplification for long distance PCR up to 22 kb for complex genomic DNA and 40 kb for less complex templates high fidelity 6 5x higher using standard nucleotide mixes than Taq polymerase and comparable to Pfu polymerase hot start mechanism using JumpStart antibody to prevent nonspecific product formation improving specificity and direct loading of PCR products onto an agarose gel without the addition of loading buffers saving time in product characterization Other DNA polymerases are also compatible with this kit however additional PCR optimization may be required to obtain comparable results 6 4 PCR Conditions PCR conditions need to be determined empirically for each set of specific primers and template It is critical to optimize PCR conditions for high specificity i e generating a single product band to avoid the need for further gel purification A hot start mechanism using JumpStart antibody which significantly improves PCR specificity has been incorporated in this kit For most applications you should only need to adjust the annealin
6. of a large number of genes has been an enormous challenge for gene function studies in the post genomics era One of the major limitations has been the lack of a pair of universal restriction enzymes for generating the cohesive ends required for directional cloning Therefore different enzymes are used for cloning different genes which restricts throughput and increases workload The Sigma Director Universal Cloning system is an ExoClone technology based solution that provides a simple rapid and universal method to facilitate the ligation of PCR products into vectors cleaved with 5 overhang producing restriction endonucleases Directionality is obtained by pairing directionally designed PCR primers with an appropriately digested plasmid vector The complete system includes a universal PCR kit and two Director Ready vector kits The PCR kit is designed for the generation of DNA inserts ready for directional cloning into any expression vector including the two vectors included with the kit It differs from conventional PCR kits in that it uses a specially formulated dNTP mix containing dATPaS dGTPas and Exonuclease Ill After PCR the cohesive 5 termini of the amplicon are produced by Exonuclease III digestion instead of being generated by traditional restriction enzyme digestions Incorporation of dA GTPasS into the amplicon provides a convenient method for protecting the amplicon from overdigestion by Exonuclease Ill The nucleotide mix
7. AGCTT target sequence 3 T T C G A A 5 Mul Ee i 5 pCGCGT target sequence 3 TGCG C A 5 Nco d T Ps 5 pCATGG target sequence 3 GGTACC 5 Nde d E nes Y 5 pATATG target sequence 3 G T A T A C 5 Nhe Ma d cd es a 5 pCTAGC target sequence 3 C GAT C G 5 Not al SE ESCH 5 pGGCCGC target sequence 3 CGCccGcG cG 5 Sall did ade e 2 5 pTCGAC target sequence 3 CAGC T G 5 Spel EE se T 5 pCTAGT target sequence 3 T GA T C A 5 Xba ae eae DES a 5 pCTAGA target sequence 3 A GAT Gm 5 Xho Pc GE x 5 pTCGAG target sequence 3 GAGC TC 5 Xma A is A 5 pCCGGG target sequence 3 GGGCCc C 5 Version Date 10 05 05 21 Director Universal Cloning System Manual APPENDIX 2 pFLAG MAC BACTERIAL EXPRESSION VECTOR INFORMATION The pFLAG MAC Vector Product Code E5644 is a 5071 bp E coli expression vector used for cloning and cytoplasmic expression of a properly inserted open reading frame as an N terminal Met FLAG fusion protein The Met FLAG fusion protein may be detected using the ANTI FLAG M2 monoclonal antibody Product Code F3165 or ANTI FLAG M5 monoclonal antibody Product Code F4042 or purified using the ANTI FLAG M2 affinity gel Product Code A2220 Note Cleavage with Xba l is sensitive to dam methylation Use a dam negative host strain Feature Map Position Xho Smal
8. Bgl II Hind III Kpn tac promoter 39 99 Metta FLAG Peptide Multiple Cloning Region Lac Binding 73 93 RBS 100 106 N26 Primer C24 Primer tac Promoter FLAG sequence 115 138 Multiple cloning region 133 179 T1T2 231 601 lac amp pFLAG MAC N26 sequencing primer 1 26 5071 bp C24 sequencing primer 223 200 Ampicillin resistance 693 1553 pBR322 ori beta lactamase 2310 f ori 2548 3011 f4 ori pBR322 ori Lac 4486 3404 Nucleotide Sequence of the Multiple cloning Region of the pFLAG Mac Expression Vector Sequence range 112 to 189 Fn II 5 ATG GAC TAC AAG GAC GAC GAT GAC AAA GTC AAG SR CTT 3 TAC CTGATG TTC CTG CTG CTA CTG TTT CAG TTC GAA MET ASP TYR LYS ASP ASP ASP ASP LYS VAL LYS LEU FLAG Peptide asi Multiple Cloning Region 255 l PS l Sma Kpn Bgl II Me Stop Stop Stop circ GAGIAAT TCC ce c GTA cic T cca GA TIC ABE EKEY ccr 3 GAG CTC TTA AGiG Gicc CA T GGA CGT CTA GAT CITA TCT ACT CGA 5 LEU ASP ASN SER ARG VAL PRO ALA ASP LEU ASP ARG Multiple Cloning Region 22 Version Date 10 05 05 Director Universal Cloning System Manual APPENDIX 3 pFLAG CMV 2 MAMMALIAN EXPRESSION VECTOR INFORMATION The pFLAG CMV 2 expression vector Product Code E7398 is a 4 7 kb derivative of the pCMV5 transient expression vector 1 for intracellular expression of N terminal Met FLAG fusion proteins in mammalian cells The promoter regulatory region of the human cytomegalovirus 2 drives transcrip
9. CA TTA AGT CTG GTT GCT AAC 3 2 REH Exonuclease III E1131 25 pal 100 u ul Molecular Biology Reagent Water W4502 1 5 ml 5 2 Additional Reagents and Equipment Not Supplied Target template DNA e Gene specific primers for sample PCR e Dedicated pipettes and aerosol resistant x pipette tips 0 5 or 0 2 ml thin walled PCR micro s centrifuge tubes e Thermal cycler P e PCR marker Product Code P9577 1 e 0 8 196 agarose gel Product Code A9539 10x Tris Borate EDTA buffer TBE Product Code T4415 GeneElute PCR Purification kit NA 1020 5 3 Storage GeneElute Gel Extraction kit Product Code NA1111 100x Tris EDTA buffer Product Code T9285 1M Tris HCl pH 8 0 Product Code T3038 0 5 M EDTA Na Product Code E7889 5 0 M NaCl Product Code 86546 LB Agar Ampicillin Plates 100 pg ml ampicillin Product Code L5667 2x JumpStart REDTaq ReadyMix Product Code P0982 All components should be stored at 20 C Avoid multiple freeze thaw cycles Version Date 10 05 05 Director Universal Cloning System Manual 6 0 GENERAL CONSIDERATIONS Please read this section thoroughly before starting your experiment 6 1 Template DNA The system is compatible with different sources of DNA template including plasmid DNA cDNA and genomic DNA However PCR performance may be affected by the complexity and purity of the DNA template 6 2 Primer Design e One set of 5 overha
10. Director Universal Cloning System User Manual a TER T tv Y a Mk ee ie en WW en S k EE 1 sn PIC E vi It age i d la E a RK 3 ws Product Codes RDC1 RDCLIG1 RDCL3 X SIGMA Director Universal Cloning System Manual 1 0 TABLE OF CONTENTS 1 0 Table of Conte nts euer eegener kees tere A cid 1 License Agreement vurn rr er EE EE KREE E RER EEE EE EE EE EE ER EE EE KREE EE EE EE EE ER ER EE EE EE EE EE ER ER EE KREE EE EE EE EE EE COREE CORE EE EE EE EE ER EE EE EE E EE EE EE EE EE EE EE ER ER EE R 2 Precautions and Disclaimer nun ek ss ennuuuhhuuunuuuuuuuuuuuuRuaRRRRRRRRRRRRARRRRRERRARSARRSRERARRSEERRSARRRRSERRRRSEERRRSARRRRSERRRRSERRRRRRERRSARRERSERRRARSERRRRRR ERE 2 2 0 Director Universal Cloning System Components eeeeeeeenennnenen nennen 3 Director Universal PCR System Product Code RDCT een een 3 Director Ready Vector Sels eebe Regia daa p EE de e HET P E debited 3 Director Ready pFLAG MAC Product Code HRDCL II 3 Director Ready pFLAG CMV 2 Product Code RDCL3 sese 3 JO INTO UCA id tas 4 SN Mee A 3 2 Featur s and Benefits dni dn tei td dre e teen hee eg 4 4 0 Director Universal Cloning System Overview REENEN EEN 5 5 0 Director Universal PCR System eeeeeeeseeseeeseeeeeeeee erre 6 5 1 Reagents Provide sess 0002 s eoe One e Dei ER D DT 6 5 2 Additional Reagents and Equipment Not Supplied
11. FLAG MAC L9158 20 ul 50 ng ul Verification primer BF 5 TGG CAA ATA TTC TGA AAT GAG C 3 P5361 50 ul 20 uM Verification primer BR 5 CTA CGG CGT TTC ACT TCT GAG T 3 P5486 50 ul 20 uM The Director Ready pFLAG CMV2 Set is used for cloning and transient intracellular expression of N terminal Met FLAG fusion proteins in mammalian cells Reagents provided in the kit are sufficient for 20 reactions Kit Component Product Number Quantity Director Ready pFLAG CMV2 L0156 20 pl 50 ng l Verification primer MF 5 AAT GTC GTA ATA ACC CCG CCC CGT TGA CGC 3 P2987 50 ul 20 uM Verification primer MR 5 TAT TAG GAC AAG GCT GGT GGG CAC 3 P3112 50 ul 20 uM 9 2 Dedicated pipettes and aerosol resistant pipette tips Competent cells such as GC5 Uni packs Product Code G3169 e LB Ampicillin agar plates 2x JumpStart REDTaq ReadyMix Product Code P0982 e 0 2 or 0 5 ml thin walled PCR microcentrifuge tubes Thermal cycler e PCR marker Product Code P9577 e 0 8 1 agarose gel Product Code A9539 e 10x Tris Borate EDTA buffer TBE Product Code T4415 9 3 Storage Additional Reagents and Equipment Not Supplied All components should be stored at 20 C Avoid multiple freeze thaw cycles Quick Link DNA ligation kit Product Code LIG2 or other T4 DNA Ligase Product Code D2886 12 Version Date 10 05 05 Director Universal Cloning System Manual 10 0 GENERA
12. L CONSIDERATIONS The ExoClone PCR product generated by this system can be cloned into a variety of commercial vectors including Sigma s FLAG series of expression vectors When using vectors other than those included with this system common precautions for preparing double digested vectors should be taken to minimize the background caused by incomplete digestion We highly recommend dephosphorylating the vector after restriction digestion in order to decrease the cloning background Since Exonuclease Ill treated PCR products contain only 5 overhangs you should choose 5 overhang producing restriction enzymes for both vector preparation and insert PCR primer design Note Please refer to Appendix I for the cleavage sites PCR primer sequences for most commonly used 5 overhang producing restriction enzymes 11 0 PROTOCOLS FOR THE DIRECTOR READY VECTOR SETS 11 1 Ligation of PCR Products Insert Since the vectors in Director Ready Vector Sets are predigested with Hind lll and Bgl Il the insert either generated by restriction digestion or via the ExoClone method must have a Hind IIl compatible site at the 5 end and a Bg Il compatible site at the 3 end We recommend that the insert generated from the control template and primers in the Director Universal Cloning System be used This will serve as a positive control for cloning efficiency Molar ratio of insert vector The optimal insert vector molar ratio for Director Universal Cloning S
13. PCR amplification Des ExoClone dNTP mix Exonuclease III PAE of PCR product Purification and quantification of TS Ill treated PCR product Ligation of PCR saat predigested vectors Vv Transformation into competent cells Vv Verification of positive clones by PCR and or sequencing Outline of Procedures for Director Universal Cloning Using ExoClone Technology Restriction Site 1 a Target DNA S 2 5 PCR dNTPoS i X Restriction Site 2 S N gt s Exonuclease III i s Double digested Vector Y Rapid Ligation and Transformation Y Directional Clones y PCR and Sequence Verification Version Date 10 05 05 Director Universal Cloning System Manual 5 0 DIRECTOR UNIVERSAL PCR SYSTEM This is a universal kit for generating Director compatible ExoClone PCR inserts Reagents provided are sufficient for 25 PCR amplifications 5 1 Reagents Provided Kit Components Product Number Quantity JumpStart REDAccuTaq Y LA DNA Polymerase D1938 62 5 ul 1 u ul 10x AccuTaq LA DNA Polymerase Buffer 500 mM Tris HCl 150 mM B0174 05ml Ammonium Sulfate pH 9 3 25 mM MgCl 1 Tween 20 i ExoClone dNTP Mix 20x E4280 62 5 ul Control PCR Template D4314 10 ul 1 ng pl Control RDC primer F with 5 phosphorylation P4986 5 p AGC TTC TCG AGA TGC CTG TTC TG 3 SE Control RDC primer R with 5 phosphorylation P5111 5 p GAT CTT CTG C
14. R product by agarose gel electrophoresis Exonuclease lll Digestion After PCR add 1 ul of Exonuclease Ill to each 50 ul PCR final concentration of 2 units tl Mix thoroughly Incubate in a water bath at 37 C for 15 minutes Purification of PCR Products If PCR results reveal a single amplification band with a reasonable yield proceed to a postreaction clean up using Sigma Aldrich PCR Purification kit Product Code NA1111 If PCR results reveal more than one band gel purification should be performed in order to obtain a specific PCR product Sigma Aldrich Gel Extraction kit Product Code NA1020 If no PCR product is observed check the PCR conditions Refer to the troubleshooting guide in this manual for assistance with optimizing PCR conditions Proceed to ligation and transformation in sections 9 11 following the Abbreviated Procedure for Experienced Users Version Date 10 05 05 9 KA D UI KA 2 a UI O Z D D a X x O Le W D 2 a UI Q o D A a W ke 2 W D t lt q Director Universal Cloning System Manual 8 0 ABBREVIATED PROCEDURE FOR EXPERIENCED USERS Note This procedure is intended for users who are familiar with PCR and have performed these reactions previously Inexperienced or first time users should refer to the complete procedures in Section 7 STEP 1 Setup PCR to generate DNA inserts Control reaction 5 ul 10x AccuTaq LA DNA Polymerase Buffer 2 5
15. e absence of an insert or PCR failure Version Date 10 05 05 15 Director Universal Cloning System Manual 11 3 6 Verification of ligation reactions optional A simple and sensitive assay using PCR can be used to check the ligation reaction if the transformation results are not satisfactory PCR should be set up as follows 8 2 ul 10 ul 0 4 ul 0 4 ul 1 0 ul H20 JumpStart REDTaq ReadyMix 2X Verification primer F 20 pmol ul or 20 uM stock Verification primer R 20 pmol ul or 20 uM stock Ligation product or serial dilution of ligation product 20 ul X number of reactions The same PCR profile as described in Section 11 3 3 can be used with this sample If the PCR results are negative it could rule out the possibility of any transformation related issues Please refer to the Troubleshooting Guide Section 12 0 for helpful suggestions 16 Version Date 10 05 05 Director Universal Cloning System Manual 12 0 TROUBLESHOOTING GUIDE Problem No PCR product is observed in ExoClone PCR amplification Multiple PCR products are observed in ExoClone PCR amplification Possible Cause A PCR component may be missing or degraded Solution A positive control should always be run to ensure components are functioning We recommend following a checklist when assembling reactions to avoid leaving out components Too few PCR cycles performed Increase the number of cycles in 3 5 cycl
16. e increments The annealing temperature may be too high Decrease the annealing temperature in 2 4 C increments The primers may not be designed optimally Confirm the accuracy of the sequence information If the primer is less than 15 nucleotides long try to lengthen the primer to 15 18 nucleotides in addition to the 5 additional bases on the 5 end If the primer has a GC content of less than 40 try to redesign the primer with a GC content of 40 60 Not enough DNA template If increasing the number of cycles has no effect repeat the reaction with a higher concentration of template Poor quality DNA template Evaluate the template integrity by agarose gel electrophoresis It may be necessary to repurify the template using methods that minimize shearing and nicking The denaturation temperature may be too high or too low Optimize the denaturation temperature by raising or lowering it in 1 C increments The denaturation time may be too long or too Short Optimize the denaturation time by increasing or decreasing it in 10 second increments The extension time may be too short Increase the extension time in 1 minute increments especially for long templates The reaction may not have enough enzyme For most applications 2 5 units reaction are sufficient We recommend that the cycling parameters be optimized before the enzyme concentration is increased In rare cases the yields ca
17. g temperature and extension time according to the T of the primers and the length Version Date 10 05 05 7 Director Universal Cloning System Manual of the template However other techniques such as using touchdown PCR may further improve the specificity of amplification 7 0 PROTOCOLS FOR THE DIRECTOR UNIVERSAL PCR SYSTEM 7 1 Setting up Control PCR The control PCR template in this system is supplied so that you may test the system performance efficiency of PCR amplification cloning etc The primers included with the control template are designed so that the amplicon will contain a full length 1485 base pair bacterial alkaline phosphatase gene GenBank 16128368 with a Hind Ill compatible restriction site at the 5 end and a Bgl II compatible restriction site at the 3 end These restriction sites allow directional insertion of the PCR product into a Hind III Bg Il double digested vector e g Sigma s Director Ready vectors We recommend that you run the control PCR along with your sample reactions Note We do not recommend the use of DMSO or formamide with JumpStart REDAccuTaq LA DNA Polymerase due to interference with the enzyme antibody complex Other co solvents salts and extremes in pH or other reaction conditions may reduce the affinity of the JumpStart antibody for the Taq polymerase and thereby compromise its effectiveness 1 Mix the following components in a 0 2 ml or 0 5 ml PCR tube The react
18. in the kit is formulated so that the amplicon terminates at a statistically determined array of 3 dA GaS sites PCR primers are designed such that the 5 termini complement the 5 overhangs of predigested cloning vectors Each of the Director Ready vector sets contains one FLAG expression vector predigested and dephos phorylated along with sequencing primers for verification of transformants These ligation ready vectors are designed to optimize efficient directional cloning of PCR products The typical cloning efficiency using these vectors is greater than 90 transformants with a positive insert in the desired orientation 3 1 Application e PCR based directional cloning of genes ORFs for expression studies 3 2 Features and Benefits e Directional cloning allows rapid gene expression e No limit on restriction enzyme selection allows rapid cloning of a large number of genes in any expression vector e Use Director Ready Vectors or create your own e Typical cloning efficiency using the Director Ready vectors is greater than 90 e Simple procedure increases throughput while decreasing hands on time 4 Version Date 10 05 05 Director Universal Cloning System Manual 4 0 DIRECTOR UNIVERSAL CLONING SYSTEM OVERVIEW Figure 1 Outline of the Procedure Select a cloning vector and restriction enzymes for double digestion Design PCR primers incorporating the desired SER sites for amplification of a target gene
19. ing efficiency Rapid ligation kits such as Quick Link Product Code LIG2 may also be used 11 2 Transformation of Ligation Product Competent cells with transformation efficiencies 210 cfu ug DNA are suitable GC5 Competent Cells Product Codes G3169 and G3044 are recommended transformation efficiency is 210 cfu ug DNA Different E coli strains such GC5 JM109 Product Code J3895 or BL21 Product Codes B2685 B2935 B3310 and B3435 competent cells can be used for transformation However since competent cells are very sensitive to temperature changes and storage conditions extreme caution should be taken to avoid freeze thaw cycles and extended storage times eg gt 6 months Follow the manufacturer s instructions for recommended transformation procedures 11 3 Verification of Transformants A variety of methods can be used to verify positive transformants including restriction digestion sequencing and PCR The following section contains a PCR based rapid verification procedure suitable for screening a large number of transformants using verification primers provided in the Director Ready Vector Kit 11 3 1 Pick colonies Optimal PCR results are obtained when small 1 mm in diameter colonies are picked from a freshly grown plate Typically the colony is resuspended in 50 ul of H20 use 9 2 ul for PCR amplification and keep the rest at 4 C for additional culturing following verification 11 3 2 PCR setup 9 2 ul Bacteria
20. ing to expired US Patent No 5 079 352 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim no right to perform any patented method and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA PRECAUTIONS AND DISCLAIMER Sigma Director Universal Cloning System is for laboratory use only not for drug household or other uses Director Director Ready ExoClone pFLAG CMV pFLAG MAC JumpStart REDAccuTaq AccuTaq Quick Link ESCORT EZMix GenElute MAT CelLytic and EZView are trademarks of Sigma Aldrich Co FLAG and ANTI FLAG are registered trademarks of Sigma Aldrich Co GC5 is a trademark of GeneChoice Inc Sigma brand products are sold through Sigma Aldrich Inc Sigma Aldrich Inc warrants that its products conform to the information contained in this and other Sigma Aldrich publications Purchaser mu
21. ion should be set up on ice 5 ul 10x AccuTaq LA DNA Polymerase Buffer 2 5 ul ExoClone dNTP Mix 20x 1 ul Control RDC Primer F 20 uM 1 ul Control RDC Primer R 20 uM 1 ul 1 4 dilution of Control PCR Template diluted to 250 pg ul 2 5 ul JumpStart REDAccuTaq LA DNA Polymerase 1 u ul 37 ul Water 50 0 pl Total Volume 2 PCR Amplification Initial denaturation 95 C 3 minutes 30 cycles Denaturation 94 C 30 seconds Annealing Extension 68 C 2 minutes Final extension 68 C 5 minutes Soak 4 C 8 Version Date 10 05 05 T 3 Director Universal Cloning System Manual Setting up Sample PCR Add the following components in 0 2 ml or 0 5 ml PCR tubes Reactions should be set up on ice 5 ul 10x AccuTaq LA DNA Polymerase Buffer 2 5 pl ExoClone dNTP Mix 20x 1 ul Forward Custom Primer 20 uM 1 ul Reverse Custom Primer 20 uM X ul Sample PCR template JumpStart REDAccuTaq LA DNA Polymerase 25W 4 u ul 38 x ul Water 50 0 ul Total Volume The PCR amplification profile will vary depending on the T value of the specific primer set and the length of the template We recommend setting the annealing temperature 3 5 C below the Tm of the primers and the extension time at 1 1 5 minute kb template length If the T of the custom primers is above 75 C use a two step PCR profile as illustrated by the control reaction 94 C for denaturation and 68 C or 72 C for annealing extension Analyze the PC
22. ivated and removed Repurify the vector to remove the alkaline phosphatase The Exo IIl incubation time may be too short Increase the Exo III treatment time to 30 minutes Ligation time may be too short The PCR product may contain impurities Increase the ligation time up to 4 hours Repurify the PCR product The competent cells may be old or not competent The molar insert vector ratio may be too low Run a positive control reaction to ensure that the competent cells are fresh and competent Do not use cells that have been frozen and thawed repeatedly Make sure the ratio was correctly calculated and increase the ratio up to 10 1 if possible If the concentration of insert DNA is too low concentrate the solution using a vacuum centrifuge The vectors were digested inefficiently by restriction enzymes Check the activity amount and incubation time of the restriction enzymes The digested vectors were treated inefficiently by alkaline phosphatase The colonies may be too old satellite colonies were picked up or culture media was brought into PCR Verify that the digested vectors were treated with alkaline phosphatase Increase the amount of enzyme and the incubation time Pick small 1 mm in diameter and fresh colonies Dilute the colonies with water instead of putting them directly into the PCR cocktail 18 Version Date 10 05 05 Director Universal Cloning System Ma
23. n be improved by increasing the enzyme concentration However if the enzyme concentration is above 5 units reaction higher background levels may be seen Mg levels may be too low Too many PCR cycles performed Optimize the magnesium concentration By reducing the number of cycles nonspecific bands may be eliminated Reduce the cycle number in 3 5 cycle increments The annealing temperature may be too low Increase the annealing extension temperature in 2 3 C increments The primers may not be designed optimally Confirm the accuracy of the sequence information If the primer is less than 15 nucleotides long try to lengthen the primer to 15 18 nucleotides in addition to the 5 additional bases on the 5 end If the primer has a GC content of less than 40 try to redesign the primer with a GC content of 40 60 Version Date 10 05 05 17 Director Universal Cloning System Manual TROUBLESHOOTING GUIDE continued Problem Products from the ExoClone PCR amplification are smeared Contamination is observed in ExoClone PCR amplification Inefficient ligation Poor transformation Poor subcloning efficiency or high background Many failed reactions in verification PCR Possible Cause Too many PCR cycles performed Solution Reduce the cycle number in 3 5 cycle increments The denaturation temperature may be low Increase the denaturation temperature in 1 C increment
24. ng producing restriction endonuclease cleavage sites should be incorporated in each 5 end of the gene specific primers Theoretically any restriction enzyme can be chosen without consideration of whether it has internal cleavage sites within a specific open reading frame since Exonuclease IIl digestion will subsequently create the appropriate cohesive ends However the restriction enzyme sites in the PCR primers must match those in the chosen vector For a list of commonly used 5 overhang producing restriction endonucleases see Appendix For the control insert a Hind III overhang compatible site 5 AGCTT 3 and a Bg ll overhang compatible site 5 GATCT 3 have been incorporated in the forward control primer and reverse control primer respectively Director Ready vectors are also compatible with inserts generated using Hind lll and Bgl Il sequences 5 DAGCTTNNN a H a LEE NNNTCTAGpS5 e Vector maps and MCS regions for the uncut parent vectors of Director Ready vectors are found in Appendices 2 and 3 A leucine residue is encoded by the last three nucleotides CTT in the Hind III cleavage site of the forward control primer e Both primers must be 5 phosphorylated This can be done during primer synthesis e Primers should be 220 bp specific target sequence plus 5 bases for restriction site to achieve adequate specificity however the length can vary depending on the sequence content of selected region The Tm GC
25. nual 13 0 REFERENCES 1 Putney S D et al A DNA fragment with an alpha phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease Ill and can be replicated in vivo Proc Natl Acad Sci USA 78 7350 7354 1981 2 Olsen D B and Eckstein F Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase exploitation for DNA sequencing Nucleic Acids Res 17 9613 9620 1989 3 Kellogg D E et al TaqStart Antibody hot start PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase BioTechniques 16 1134 1137 1994 4 Don R H et al Touchdown PCR to circumvent spurious priming during gene amplification Nucleic Acids Res 19 4008 1991 Version Date 10 05 05 19 Director Universal Cloning System Manual 14 0 RELATED PRODUCTS e Expression vectors containing FLAG c Myc or MAT tags e LB broth EZMix powder Product Code L7658 and EZMix LB Agar powder Product Code L7533 e GenElute plasmid purification kits and endotoxin free plasmid purification kits e JumpStart REDAccuTaq LA DNA polymerase Product Code D1313 e Custom Oligonucleotides Sigma Genosys offers a complete line of custom oligonucleotides for your research needs For more information call 800 234 5DNA or visit our website www sigma aldrich com e Cell culture media e ESCORT family of transfection reagents Prod
26. s The extension time may be too long Decrease the extension time in 1 2 minute increments Too much enzyme in the reaction mix For most applications 2 5 units reaction are sufficient However this concentration may be too high for some applications We recommend optimizing the cycling parameters first as described above then if necessary incrementally reduce the enzyme concentration to determine the optimal concentration Mg concentration may be too high Optimize the magnesium concentration The template concentration may be too high Reduce the concentration of the template in the reaction Contamination usually results in extra bands or smearing We recommend running a water control in place of the DNA template for every set of reactions to determine if the reagents used in the PCR are contaminated with a template from a previous reaction When performing PCR directly on phage plaques or bacterial colonies failure to isolate single plaques or colonies will produce multiple bands A ligation reaction component may be missing or degraded A positive control should be run to ensure components are functional The primers were not designed properly Verify that the primer design correctly included the restriction enzyme site Primers must be 5 phosphorylated in order to function The PCR product may contain impurities Repurify the PCR product Alkaline phosphatase was not inact
27. sssssssssssese 6 e S otofage coto Ito rien etes ete edema 6 BU General Considerations eet ee EES eR Ras gane EUER e eR EE 7 6 1 Template DNA ti da diia perite e Aa Eae ine PERRA CURE ERU ERR de te qed aid 7 6 2 gt PRIME DESIQM ete 7 6 3 DNA Polymerase lt 3 idt tiere A ERI Re ed 7 604 Ee ee o ERSTER ETE SENE RU 7 7 0 Protocols for the Director Universal PCR System eeeseeeeeeeeneneenennenenn nennen 8 fA Setting up Control POR edi cce ca rti edere eI EENEG 8 7 2 Setting p Sambple POR aceite etn e i ei ede ee adnate 9 7 3 Exonuclease lll DigeStion cecccceccecsececsesceeeenecceseensnneceeecceceenaneeeesbenseaeeneeeeeesneneesestaneeeteenanes 9 7 4 Purification of PCR Products nemen nennen neret er Aa nnne nter tenens 9 8 0 Abbreviated Procedure for Experienced Users use EEN EEEEEE REENEN 10 9 0 Director Ready Vector Sets esseeeeeeeeeeee ecc nennen nnn i nannte nnne nnns inan nenne innuens 12 9 1 Reagents Provided EE 12 9 2 Additional Reagents and Equipment Not Supplied ooococinnnnnocccnnnnnnncccononccccnnccncnnanonccnnnnnnns 12 NS senesi iritira ened sien dike davies hie AEEA nein chars 12 10 0 General Considerations orien ii ge 13 11 0 Protocols for the Director Ready Vector Sets seeeseeeeeenenene nennen nnns 13 EET Ligation of PCR deele 13 11 2 Transformation of Ligation Product sse nennen
28. st determine the suitability of the product s for their particular use Additional terms and conditions may apply Please see reverse side of the invoice or packing slip 2 Version Date 09 17 10 Director Universal Cloning System Manual 2 0 DIRECTOR UNIVERSAL CLONING SYSTEM COMPONENTS Director Universal PCR System Product Code RDC1 e JumpStart REDAccuTaq LA DNA Polymerase e 10x AccuTaq LA DNA Polymerase Buffer e ExoClone dNTP Mix 20x e Control PCR Template e Control RDC primer F with 5 phosphorylation e Control RDC primer R with 5 phosphorylation e Exonuclease lll e Molecular Biology Reagent Water Storage Conditions Store all components at 20 C Avoid multiple freeze thaw cycles Director Ready Vector Sets Director Ready vectors are pre digested with Hind IIl and Bgl Il Director Ready pFLAG MAC Product Code RDCLIG1 e Director Ready pFLAG MAC Expression Vector e Verification primer BF e Verification primer BR Storage Conditions Store all components at 20 C Avoid multiple freeze thaw cycles Director Ready pFLAG CMV 2 Product Code RDCL3 e Director Ready pFLAG CMV 2 Expression Vector e Verification primer MF e Verification primer MR Storage Conditions Store all components at 20 C Avoid multiple freeze thaw cycles Version Date 10 05 05 3 Director Universal Cloning System Manual 3 0 INTRODUCTION The cloning and expression
29. tion of FLAG fusion constructs The pFLAG CMV 2 expression vector is a shuttle vector for E coli and mammalian cells The SVAO origin of replication results in most efficient replication of the vector in COS cells Verification Primers MF and MR are equivalent to the CMV 30 and CMV 24 sequencing primers References 1 Andersson S et al J Biol Chem 264 8222 8229 1989 2 Thomsen D R et al Proc Natl Acad Sci USA 81 659 663 1984 Feature Map Position Not Clal EcoR V Sall BamH Hind IIl EcoR I Bgl II Kpnl Xba Sma CMV promoter 166 916 T FLAG Peptide Multiple Cloning Region CMV 30 sequencing primer 825 854 f4 intragenic region CMV Promoter Translational initiation 932 934 FLAG sequence 935 958 CMV 30 Primer Multiple cloning region 956 1022 pFLAG CMV 2 SE SH hGH poly A 1023 1642 pBR322 ori beta lactamase 4 7 kb CMV 24 sequencing primer 1080 1103 hGH poly A SV40 ori 1661 2005 pBR322 ori beta lactamase 2908 4092 SV40 ori f intragenic region 4227 4679 Nucleotide Sequence of the Multiple cloning Region of the pFLAG CMV 2 Expression Vector Hind III Not EcoR I Yv v Y 5 ATG GAC TAC AAA GAC GAT GAC GAC AAG AAG CTT GCiG GCC GCG AAT TCA 3 TAC CTGATG TTT CTG CTA CTG CTG TTT TTC GAA CGC CGGiCGC TTA AGT MET ASP TYR LYS ASP ASP ASP ASP LYS LYS LEU ALA ALA ALA ASN SER FLAG Peptide Multiple Cloning Region Ear ene Kpn Sal e
30. uct Code E9770 L6037 L3037 L3287 E9778 e CelLytic B cell lysis reagent standard strength Product Code B7435 e CelLytic B cell lysis reagent 2x concentrate Product Code B7310 CelLytic M protein extraction reagent Product Code C2978 CelLytic MT protein extraction reagent Product Code C3228 ANTI FLAG M2 monoclonal antibody unconjugated Product Code F3165 ANTI FLAG M2 affinity gel Product Code A2220 FLAG peptide Product Code F3290 e FLAG immunoprecipitation kit Product Code FLAGIPT 1 High throughput co immunoprecipitation kit Product Code HTCOIP 1 e Co immunoprecipitation control plasmid kit Product Code COIPP e Co immunoprecipitation detection kit Product Code COIPD e EZview Red Protein A Affinity Gel Product Code P6486 EZview Red ANTI FLAG M2 Affinity Gel Product Code F2426 20 Version Date 10 05 05 Director Universal Cloning System Manual APPENDIX 1 CLEAVAGE SITES PCR PRIMER SEQUENCES FOR MOST COMMONLY USED 5 OVERHANG PRODUCING RESTRICTION ENZYMES Restriction Endonuclease Cleavage Site PCR Primer Design p phosphate BamH erg de E Se 5 pGATCC target sequence 3 C CT A GG 5 Bgl II ce qud e 2 5 pGATCT target sequence 3 T C TA G A 5 Cla asd be P 2 5 pTCGAT target sequence 3 T AGC TA 5 EcoR ee NS a 5 pAATTC target sequence 3 C T T A A G 5 Hind III CREE EA SEH il 5 p
31. ul ExoClone dNTP Mix 20x 1 ul Control RDC Primer F 20 uM 1ul Control RDC Primer R 20 uM 1 ul 1 4 dilution of Control PCR Template diluted to 250 pg ul 2 5 ul JumpStart REDAccuTaq LA DNA Polymerase 1 u ul 37 ul Water 50 ul Total Volume Sample reaction 5 ul 10x AccuTaq LA DNA Polymerase Buffer 2 5 ul ExoClone dNTP Mix 20x 1 yl Forward Custom Primer 20 uM 1 yl Reverse Custom Primer 20 uM X ul Custom PCR Template 2 5 ul JumpStart REDAccuTaq LA Polymerase 1 u ul 38 x ul Water 50 ul Total Volume 10 Version Date 10 05 05 Director Universal Cloning System Manual STEP 2 PCR amplification Protocols optimized for the control template primers and the specific template primers are described below Control PCR Initial denaturation 95 C 3 minutes 30 cycles Denaturation 94 C 30 seconds Annealing Extension 68 C 2 minutes Final extension 68 C 5 minutes Soak 4 C Sample PCR see Section 7 2 STEP 3 STEP 4 STEP 5 STEP 6 STEP 7 STEP 8 Exonuclease Ill digestion Add 1 ul of Exonuclease III to each PCR and incubate at 37 C for 15 minutes Purification of PCR product Use a PCR clean up kit or a gel extraction kit if PCR shows multiple bands Optional quantification of insert DNA Analyze the PCR products by agarose gel electrophoresis Ligation of insert DNA and vector PCR products may be rapidly ligated into Director Ready vectors usingT4 DNA Ligase
32. ystem is 5 10 1 which is higher than the conventional 3 1 ratio used in cohesive end ligation Extreme insert size or large vectors could also alter the molar ratio for optimal ligation performance f mass of insert size of vector Equation molar ratio of insert vector x mass of vector size of insert Ligase The ligation reactions should be performed using Sigma s T4 DNA Ligase Product Code D2886 or another qualified ligation product Follow the manufacturer s instructions The following protocol is provided as an example 1 Thaw the quantified control sample insert DNA the chosen Director Ready Vector Kit and the ligase on ice Thaw the other tubes at room temperature Vortex each tube briefly and then spin them down for a few seconds Once thawed keep each reagent on ice 2 Set up the ligation reaction x ul H20 _ ul Control insert or sample insert nl Director Ready vector or custom vector 50 ng ul Ligation buffer 19 ul Total Volume Version Date 10 05 05 13 Director Universal Cloning System Manual 3 Add 1 ul of T4 DNA ligase Mix thoroughly and incubate at room temperature for at least 60 minutes Note Typically 60 minutes is long enough for ligations performed with optimal insert vector ratios A longer ligation time may be required if reactions are performed with suboptimal insert vector ratios In this situation an optimization experiment should be performed to determine optimal clon
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