PCR实验报告(分子生物学实验)

PCR

Lin Chengyu Bio 04 2010030007; Cooperator: Yuan Xiaowen

Experiment date: 2012-03-22 Submit Date: 2012-03-23

1Introduction

1.1Background information

PCR (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 gene

cloning, 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 principles

One typical PCR cycle consists of three steps: denaturation, annealing and extension.

(1)Denaturation

Figure 1 Step 1: denaturation

As shown in Figure 1, the target double-strand DNA can be separated into

single-strand DNA under high temperature (about 50 ℃).

(2)Annealing

Figure 2 Step 2: annealing

As 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)Extension

Figure 3 Step 3: extension

As 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 DNA

strand complementary to the target DNA will be synthesize at the end of 3’ ends of

the primer until temperature rise to denature the double strand again.

2Experiment Operation

2.1Material

(1)pCMV-Myc-T10 (SIPAR), 10 ng;

Figure 4 Plasmid profile (pCMV – Myc)

(2)Forward and reverse primer, 1 μM;

Figure 5 Forward primer

Figure 6 Reverse primer

(3)DNA polymerase: r Taq, 5 U / μl.

2.2Chemicals and apparatus

2.2.1Solutions

Table 1 Solutions

2.2.2Apparatus

(1)Pipettes;

(2)PCR systems;

(3)Eppendorf tubes;

(4)Centrifuge;

(5)Gel electrophoresis apparatus;

(6)UV gel imaging system;

(7)Biodev PCR purification kit.

2.3Procedure

2.3.1PCR

(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) into

the 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 as

well;

(4) Add 4 μl 3×Loading buffer, 4 μl ddH2O and 4 μl PCR product to a piece

of sealing film, and mix with pipette;

(5) Carefully remove the comb, place the mold in the electrophoresis

chamber, 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 4

μl of 1kb DNA ladder into the slot beside the sample;

(7) Start electrophoresis immediately samples loading at 100 V, and stop

when 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 UV

at 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 rpm

for 30 sec. Discard the waste solution in the collection tube;

(3)Add 450 μl Washing buffer to the mini-spin column, and centrifuge at

12,000 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 a

new Eppendorf tube, and incubate for 1 min. Then centrifuge at 12,000 rpm

for 1 min.

(7)Preserve the product under -20 ℃.

3Result

Figure 7 PCR product agarose gel electrophoresis:

Lane I – Product of Y uan

Lane II – Product of Lin

The target gene, together with restriction enzyme recognition sequence added in the

primer, is about 0.9 kb long, which corresponds with the band in Figure 7.

4Conclusion

The 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.