PCRLin Chengyu Bio 04 2010030007; Cooperator: Yuan XiaowenExperiment date: 2012-03-22 Submit Date: 2012-03-231Introduction1.1Background informationPCR (polymerase chain reaction) is a technique for amplifying DNA sequences in vitro.It was invented by Kary Mullis and his colleagues in 1985. It is widely used in genecloning, mutation, sequencing and detection.1.2Objectives(1)Learn the principle and method of PCR (Polymerase Chain Reaction).(2)Comprehend the significance of PCR technique in DNA manipulation.1.3Major principlesOne typical PCR cycle consists of three steps: denaturation, annealing and extension.(1)DenaturationFigure 1 Step 1: denaturationAs shown in Figure 1, the target double-strand DNA can be separated intosingle-strand DNA under high temperature (about 50 ℃).(2)AnnealingFigure 2 Step 2: annealingAs shown in Figure 2, when temperature is lower, the forward and reverse primers will bind to the single strand DNA, which are about 20 nt long, designed to be complementary to the both end of the target gene and often has restriction enzyme recognition sites at the 5’ ends.(3)ExtensionFigure 3 Step 3: extensionAs shown in Figure 3, when temperature rise to about 72 ℃, which is the optimal temperature of Taq, one kind of DNA polymerase working in high temperature. The discovery of Taq DNA polymerase is fundamental to PCR. As a result, new DNAstrand complementary to the target DNA will be synthesize at the end of 3’ ends ofthe primer until temperature rise to denature the double strand again.2Experiment Operation2.1Material(1)pCMV-Myc-T10 (SIPAR), 10 ng;Figure 4 Plasmid profile (pCMV – Myc)(2)Forward and reverse primer, 1 µM;Figure 5 Forward primerFigure 6 Reverse primer(3)DNA polymerase: r Taq, 5 U / µl.2.2Chemicals and apparatus2.2.1SolutionsTable 1 Solutions2.2.2Apparatus(1)Pipettes;(2)PCR systems;(3)Eppendorf tubes;(4)Centrifuge;(5)Gel electrophoresis apparatus;(6)UV gel imaging system;(7)Biodev PCR purification kit.2.3Procedure2.3.1PCR(1)Add all 7 kinds of materials or solutions into PCR tubes following Table 2;Table 2 50 µl system for PCR(2)Put the tubes into the instrument for PCR, set program as Table 3;(3)Take out the tubes when program stops.2.3.2Agarose gel electrophoresis(1) During PCR, place a clean electrophoresis mold on a horizontal surface,carefully insert a comb (15 µl slots) into the mold;(2) Pour the agarose gel solution (contains 1.0 g agarose in 100 ml TAE) intothe mold gently to avoid bubbles. The height of gel shall be slightly higher than the black line on the side face;(3) Wait for approximately 30 min for solidification, when PCR complete aswell;(4) Add 4 µl 3×Loading buffer, 4 µl ddH2O and 4 µl PCR product to a pieceof sealing film, and mix with pipette;(5) Carefully remove the comb, place the mold in the electrophoresischamber, and add 1×TAE buffer in order to cover the gel to a depth of approximately 1 mm;(6) Load 15 µl solution into the slots of the gel: one slot for one tube. Load 4µl of 1kb DNA ladder into the slot beside the sample;(7) Start electrophoresis immediately samples loading at 100 V, and stopwhen Bromophenol blue is 2/3 gel length from the starting line;(8) Place the gel into EB working solution for 20 min, and observe under UVat 310 nm. The red fluorescent bands show where DNA is and their darkness show the quantity. Then take photograph for records;2.3.3PCR product purification(1)Add 50 µl PCR product to 400 µl Binding buffer, and mix with pipette;(2)Transfer the solution to the mini-spin column, and centrifuge at 12,000 rpmfor 30 sec. Discard the waste solution in the collection tube;(3)Add 450 µl Washing buffer to the mini-spin column, and centrifuge at12,000 rpm for 30 sec. Discard the waste solution in the collection tube;(4)Repeat Step 3 once more;(5)Centrifuge at 12,000 rpm for 2 min to get rid of ethanol;(6)Add 40 µl ddH2O to the center of the mini-spin column, put the column in anew Eppendorf tube, and incubate for 1 min. Then centrifuge at 12,000 rpmfor 1 min.(7)Preserve the product under -20 ℃.3ResultFigure 7 PCR product agarose gel electrophoresis:Lane I – Product of Y uanLane II – Product of LinThe target gene, together with restriction enzyme recognition sequence added in theprimer, is about 0.9 kb long, which corresponds with the band in Figure 7.4ConclusionThe PCR product contains the target gene we want to amplify in the template, and its quantity is enough for the following operation.5Reference【1】Liu Jinyuan, Zhang Shuping, Wu Yaoting, Introduction of Molecular Biology Experiment.。