| PCR扩增DNA | | 点击： 作者： 来源： 时间： 1970-01-01 本站论坛 |
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The basic PCR is applied to amplify those DNA fragments whose sequence has been known. The reaction cycle comprises a 95℃ step to denature the duplex DNA, an annealing step of around 37℃～55℃ (the actual temperature depends on the primer lengths and sequences.) to allow the primers to bind and a 72℃ polymerization step. Taq DNA polymerase is usually used as thermostable DNA polymerase in PCR, which still survives the denaturation step of 95℃. In addition, 30 cycles are used in the basic PCR.
【Materials】
1.Apparatus:
PCR thermal cycler，Table high-speed centrifuger，Pipettor，Eppendorf tubes sterilized by high pressure，Electrophoresis apparatus.
2. Reagent:
（1）Template DNA，0.1µg/µl：: Pick up single colony and suspend it in 50µl lysis solution( 1%TritonX-100，20mmol/LTris.Cl PH 8.2, 2mmol/L EDTA), put it in a warm bath of 95℃ for 10 minutes, then centrifuge at 10000 rpm for 5 minutes and remove the supernatant of 1µl to be used to PCR whose total reaction volume is 50µl.
（2）Purchase Taq DNA polymerase（5U/µl），10×amplification buffer，dNTPs solution containing all four dNTPs（1mmol/L）
（3）Primers，100pmol/L：Design and synthesis primers each complementary to one end of the target DNA.
【Procedures】
1． Preparation of PCR reaction solution
（1）In a sterile 0.5ml Eppendorf tube, mix in the following order:
10×amplification buffer 10µl
solution of four dNTPs 4µl
primer 1 1µl
primer 2 1µl
DNA template 2µl（10ng）
Taq DNA polymerase 1µl（2.5U）
Add water to terminal volume of 50µl
（2）Pellet the bottom of the Eppendorf tube with finger gently in order to mix the solution well. Then the solution is collected by being put into the table high-speed centrifuger.
（3）Overlay the reaction mixtures with 50µl of paraffin.
2．The process of PCR
The Eppendorf tubes containing the reaction mixtures are put into the PCR thermal cycler, the reaction mixtures are kept at 95℃ for 5 minutes to denature DNA fully., Next in turns of denaturing at 95℃ for 1 minute ,annealing at 55℃ for 45 seconds and extending at 72℃ for 1 minute, repeat for 30 cycles. After these cycles are finished, the reaction mixtures are extended at 72℃ for a further 8 minutes. Finally，take out the sample tubes and store at -4℃.
【Results】
Withdraw the samples and analyze them by electrophoresis through an agarose or polyacrylamide gel. Stain the gel with ethidium bromide to visualize the DNA. For the instruction in further details, please see 《Experiment 6 》.
【Discussion】
1.If PCR produces some nonspecific amplification, it may be necessary to optimize the reaction conditions. The parameters to be varied include the annealing temperature, the annealing time and the Mg2 concentration.
2.PCR has high specificity，so the concentration of the primes、Taq polymerase and dNTP should not be too high.
3.Design the primes rationally. PCR primers generally range in length from 18-30 bases. Primers should contain 40%～60% G＋C and care should be taken to avoid sequences which would produce internal secondary structure. The 3ˊends of the primers should not be complementary to avoid the production of primer-dimers in the PCR reaction. Avoid three G or C nucleotides in a row near the 3ˊend of the primer. Ideally, both primers should have similar G＋C content so that they anneal to their complementary sequences at similar temperatures. Additionally, the sequence of the primers can also include regions at the 5ˊend which can be useful for downstream applications. For example, restriction enzyme sites can be placed in the primer pair design if the desired PCR product is to be subsequently cloned.
【Advisement after experiment】
What is the effect of PCR?
上一篇：基因的PCR扩增（聚合酶链式反应） 下一篇：PCR反应条件的选择及优化
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