文档介绍:PCR基因扩增
模板
PCR反应必须以DNA为模板进行扩增,单链分子、双链分子、线状分子或环状分子均可以作为模板 DNA (线状分子比环状分子的扩增效果稍好)
模板的数量和纯度均会影响PCR结果:一般反应中的模板数量为102this sequence
Double stranded
DNA template
5’
3’
5’
3’
PCR Cycle 1
Primers
Taq
Taq
Double stranded
DNA template
5’
5’
3’
3’
PCR Cycle 1
Denaturation
95 ℃
strands separate
Primers
Taq
Taq
Taq polymerase is thermostable
3’
5’
5’
3’
Annealing
~55 ℃ Primers bind
Taq
Taq
Forward primer anneals to lower strand
Reverse primer anneals to upper strand
PCR Cycle 1
3’
5’
5’
3’
Annealing
~55 ℃
Taq binds
Taq
Taq
PCR Cycle 1
3’
5’
5’
3’
Extension
72 ℃
Taq copies DNA strand dNTPs
Taq
Taq
Taq synthesises DNA in the 5’ to 3’ direction
PCR Cycle 1
3’
5’
5’
3’
Taq
Taq
Taq synthesises DNA in the 5’ to 3’ direction
PCR Cycle 1
Extension
72 ℃
Taq copies DNA strand dNTPs
3’
5’
5’
3’
Taq
Taq
Taq synthesises DNA in the 5’ to 3’ direction
PCR Cycle 1
Extension
72 ℃
Taq copies DNA strand dNTPs
3’
5’
5’
3’
Taq synthesises DNA in the 5’ to 3’ direction
PCR Cycle 1
Taq
Taq
Extension
72 ℃
Taq copies DNA strand dNTPs
3’
5’
5’
3’
5’
3’
5’
3’
End cycle 1
PCR CYCLE 1
3’
5’
5’
3’
5’
5’
3’
3’
PCR Cycle 2
Denaturation
95℃
strands separate
3’
5’
5’
3’
5’
5’
3’
3’
Annealing
~55 ℃
Primers bind
PCR Cycle 2
Extension
72 ℃
Taq copies
DNA strand
PCR Cycle 2
Two single strands
of correct length
PCR Cycle 2
End cycle 2
End cycle 3
End cycle 4
Following n cycles of PCR there will be No(1+Y)n copies
No initial number of targets
Y efficiency of PCR reaction
n cycle number
三、仪器、材料与试剂
PCR热循环仪
移液枪(配套枪头)
DNA模板(质粒R-pGEX-4T-1,R指代外源基因)
dNTP(TaKaRa)
10×PCR缓冲液(TaKaRa)
25mmol/L MgCl2 (TaKaRa)
引物1、引物2(5pmol/μL)
引物1:5’-CGGAATTCATGGACGGTCAGA-3’
引物2:5’-GCGTCGACTCATTCAGATTTGCG-3’
Taq DNA聚合酶(TaKaRa)
四、实验步骤
PCR反应体系的配制, Eppendorf管内配制25μL反应体系,按下表准确加入各反应物
反应物
体积(μL)
10×PCR缓冲液
25mmol/L