PCR* Kits For DNA Ladder Assay
Contents
1. 5 Incubate reactions at 10 C for at least 10 min then add 0 5 ul of T4 DNA Ligase Make sure the ligase is thoroughly suspended before pipetting 6 Incubate reactions at 16 C for 12 16 hours overnight Adaptor ligated DNA may be stored at 20 C until needed for PCR 7 Calculate the concentration of DNA in your ligation reactions using the following formula Quantity of DNA in ng Volume of Ligation Mix ul Volume of DNA added in step 2 ul From this value in ng ul calculate the volume necessary to obtain the amount of adaptor ligated DNA for PCR The amount of adaptor ligated DNA required for PCR may vary we have found the optimal range to be 50 150 ng However we have obtained ladders with as little as 10 ng of DNA albeit with much lower intensity 8 For each LA PCR sample prepare a PCR mixture as follows per rxn 2X LA PCR Buffer 25 ul containing chemicals enhancer stabilizer and dNTPs PCR grade H 2 O 9 ul Adaptor Primer 10X Sul Adaptor ligated DNA 50 150 ng 10 ul Taq DNA Polymerase 5 U ul lul Total volume 50 ul It is necessary to use one of Hot start PCR methods which can be achieved by heating above mixture without Taq Pol at 72C for 2min and then add Taq Pol 9 Overlay with 50 ul of mineral oil if necessary PROCEDURE 10 PCR thermocycle profile Reaction profiles will need to be optimized according to the machine type and needs of user Please take note that temperatu2. 2 Isolate the kits from any sources of contaminating DNA especially amplified PCR product 3 Store all of components in detection kit at 2 8 C Under these conditions components of the kit are stable for 1 year 4 Donot mix LA PCR kit components that are from different lots Each lot is optimized individually REFERENCES Arends M J Morris R G amp Wyllie A H 1990 Apoptosis the role of the endonuclease Am J Pathol 136 593 608 Ausubel F M Brent R Kingston R E Moore D D Sidman J G Smith J A amp Struhl K 1994 in Current Protocols in Molecular Biology Greene Publishing Associates and John Wiley amp Sons Inc NY Vol 1 Ch 2 2 Barnes W M 1994 PCR amplification of up to 35 kb DNA with high fidelity and high yield from l bacteriophage templates Proc Natl Acad Sci USA 91 2216 2220 Cheng S Fockler C Barnes W M amp Higuchi R 1994 Effective amplification of long targets from cloned inserts and human genomic DNA Proc Natl Acad Sci USA 91 5695 5699 Hale A J Smith C A Sutherland L C Stoneman V E Longthorne V Culhane A C amp Williams G T 1996 Apoptosis molecular regulation of cell death Eur J Biochem 237 884 Iwata M 1996 Regulation of thymocyte apoptosis and positive selection Nippon Rinsho 54 1753 1759 Kellogg D E Rybalkin I Chen S Mukhamedova N Vlasik T Siebert P amp Chenchik A 1994 TaqStart Antibod
3. PCR Kits For DNA Ladder Assay Cat APO DNAI1 INSTRUCTION MANUAL The Polymerase Chain Reaction PCR process is covered by patents owned by Hoffman LaRoche Use of the PCR process requires a license A license for diagnostic purposes may be obtained from Roche Molecular System A license for research may be obtained by the purchase and the use of authorized reagents and DNA thermocyclers from the Perkin Elmer Corporation or by negotiating a license with Perkin Elmer This product is intended for research use only and not for diagnostic purposes qi Maxim Biotech Inc 780 Dubuque Avenue Tel 800 989 6296 So San Francisco CA 94080 Fax 650 871 2857 U S A Website http www maximbio com Feb 2000 APO M10000001 INTRODUCTION The DNA Ladder Assay PCR Kit LA PCR Cat APO DNA1 is designed for the detection of nucleosomal ladders in apoptotic cells Based on ligation dependent PCR Staley et al 1997 this kit enables researchers to visualize ladders that are undetectable by other methods Moreover the PCR assay is semiquantitative allowing comparison of the relative extent of apoptosis in different samples During apoptosis cellular endonucleases cleave genomic DNA between nucleo somes producing fragments whose lengths vary by multiples of 180 200 bp Wyllie 1980 Arends et al 1992 When resolved by agarose EtBr gel electro phoresis these DNA fragments appear as a nucleosomal ladder a widely recognize
4. d hallmark of apoptotic cell death Hale et al 1996 Traditionally such ladders have been assayed by electrophoresing genomic DNA samples directly on agarose EtBr gels However in biological systems in which only a small percentage of cells are apoptotic or in which apoptosis is occurring asynchro nously genomic DNA ladders may not be visible or appear as a smear The PCR assay uses PCR to specifically amplify the nucleosomal ladder making it easier to visualize Figure shows how the DNA Ladder Assay PCR works The first step is the ligation of dephosphorylated adaptors com posed of a 12 mer and a 27 mer to the ends of the DNA fragments generated during apoptosis In mammalian cells such fragments generally have 5 phosphorylated blunt ends Staley et al 1997 thus only the 27 mer is ligated to the DNA fragments When the mixture of ligated DNA fragments is heated the 12 mer is released Next the 5 protruding ends of the molecules are filled in by a thermostable DNA polymerase The 27 mer then serves as a primer in a PCR in which the fragments with adaptors on both ends are exponentially amplified The resulting nucleosomal ladder can be easily visualized on an agarose EtBr gel High sensitivity and low background Because of its high sensitivity the DNA Ladder Assay PCR reveals ladders that may be undetectable by the conventional method The nucleosomal ladder could not be detected when same genomic DNA as from Figure was directly el
5. ectrophore sed on the gel data not shown The DNA Ladder Assay PCR assay is semiquantitative By comparing the numbers of PCR cycles needed to detect ladders in two samples the relative amount of apoptosis occurring in each sample can be estimated The DNA Ladder Assay PCR Kit includes optimized reagents for performing this assay The kit includes T4 DNA Ligase buffers PCR primers controls anda complete User Manual However the thermostable DNA polymerase is not included continue adaptor genomic DNA fragment adaptor 27 mer 5 3 HO m OH P OH HO OH HO E OH HO P HO M O H 12 mer 3 5 Ligate adaptor to DNA fragments v BE E Heat to release 12 mer M EE ee Fill in ends with thermostable DNA polymerase v ae gt lt Amplify by PCR 30 35 cycles y ee ane n ee ee ee ee eee eee eee eee eee a E nnnm gt is a Resolve amplified fragments on agarose EtBr gel kb 1 000 800 600 400 200 Figure 1 Schematic diagram of the PCR assay For the gel shown here apoptosis was induced in Keratinocyte cells by incubation with 10uM CPT for 6 hr Genomic DNA was isolated from uninduced and induced Keratinocytes and was used in the PCR assay according to the User Manual 30 PCR cycles The optimal number of PCR cycles must be determined for each genomic DNA sample 11 ul of each reaction was electrophore
6. nucleotide 5 GAC GTC GAC GTC GTA CAC GTG TCG ACT 3 ADAPTORPRIMER 27 nt oligonucleotide 5 GAC GTC GAC GTC GTA CAC GTG TCG ACT 3 Materials Not Included Taq DNA Polymerase Perkin Elmer or it s Agarose Gel equivalent recommended Timer Phenol Electrophoresis Apparatuus Chloroform TE Buffer Forceps orit s equivalent recommended TBE Buffer Oven s or incubator s Set Tm to 37 C amp 42 C Pipettes and tips P1000 P200 and P20 NOTE SPIN ALL TUBES BEFORE USING PROCEDURE 1 Extract genomic DNA from tissue or cells of interest Resuspend DNA in PCR grade H 20 or TE Note RNase treatment is not necessary as residual RNA will not interfere with this procedure However treatment with DNase free RNase and reprecipitation is necessary if you are using UV spectrophotometry OD 260 to quantify DNA Be sure to wash DNA thoroughly to remove salts that will inhibit the ligation reaction 2 Mix 0 5 ug of genomic DNA with Ligation Buffer Adaptor etc 10X Ligation Buffer Sul Genomic DNA or Control DNA 10X Sul Adaptors 10X Sul ddH20 34 5 ul If you use more or less than 0 5 ug of genomic DNA you will need to adjust the volume of Ligation Mix Overlay sample with mineral oil 3 Heat reaction mixture to 55 C for 10 min flick tube to mix 4 Allow the adaptor oligonucleotides to anneal by cooling to 10 C over approximately 1 hr Note This gradual temperature ramp down may be programmed into a thermal cycler for convenience
7. re variations occur between different thermocyclers therefore the annealing temperature in the sample profile below is given as a range It will be necessary to determine the optimal temperature for your individual thermocycler An example of a time temperature profile for the positive control PCR reaction optimized for Perkin Elmer machine types 480 and 9600 is provided below Temperature Time 72 C 94 C 94 C 70 C 72 C 20 C 11 Electrophorese 10 15 ul of each above reaction is mixed with loading buffer and loaded on a 1 5 2 agarose EtBr gel TROUBLESHOOTING 1 LA PCR AMPLIFICATION Observation 1 1 No signal or no ladder 1 2 during amplification even using positive control provided in kit Too many nonspecific bands l la 1 2a 1 2b 1 2c Possible Cause The annealing temperature in thermocycler is too high Dominant primer dimers The annealing temperature in thermocycler is too low Pre PCR mispriming DNA is interfering with LA PCR l la 1 2a 1 2b 1 2c Recommended Action Decrease PCR annealing tempera ture 3 5 C gradually Use any one of Hot Start PCR procedures Increase PCR anneal temperature 3 5 C gradually Use any one of Hot Start PCR procedures Clean DNA with Phenol Chloroform PRECAUTIONS AND STORAGE Storage 1 Store all LA PCR Kit components at 20 C Under these conditions components of the kit are stable for 1 year
8. sed on a 1 5 agarose EtBr gel Lane 1 uninduced Lane 2 induced apoptotic Lane M 100 bp DNA ladder markers GENERAL CONSIDERATION e For genomic DNA isolation we recommend the protocol described in Ausubel et al 1994 or purchase Maxim s DNA isolation kit Cat EXT 0001 For best results use 0 5 ug of genomic DNA for each ligation We recommend that you do not use less than 0 25 ug of genomic DNA The PCR conditions in this protocol have been optimized using a Perkin Elmer DNA Thermal Cycler 2400 The actual number of cycles performed will vary and must be determined empirically for each genomic DNA sample e Add enzymes to reaction mixtures last and thoroughly incorporate the enzyme by gently pipetting the reaction mixture up and down Hot start PCR is highly recommended for LA PCR which can be achieved by adding Taq Pol at 72C MATERIALS AND REAGENTS APO DNA1 50 test DNA Ladder Assay PCR LA PCR Kit Each PCR Amplification Detection Kit contains the following reagents and materials Catalog No Kit Component Amount Storage N A 10X Ligation Buffer 250 ul N A T4 DNA Ligase 400 units ul 25 ul DNA1 ADP 10X Adaptors 250 ul DNA1 C001 10X Pos Control DNA 50 ul DNA1 B001 2X LA PCR Buffer 1250 ul DNA1 P001 10X Adaptor PCR Primers 250 ul MRB 0014 DNA M W Marker 100bp Ladder 100 ul N A ddH O DNase free 2 0 ml Instruction Manual 1 copy ADAPTOR 12 nt oligonucleotide 5 AGT CGA CAC GTG 3 27 nt oligo
9. y Hotstart PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase BioTechniques 16 1134 1137 Staley K Blaschke A J amp Chun J 1997 Apoptotic DNA fragmentation is detected by a semiquantitative ligation mediated PCR of blunt DNA ends Cell Death Diff 4 66 75 Wyllie A H 1980 Glucocorticoid induced thymocyte apoptosis is associated with endogenous endonuclease activation Nature 284 555 556
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