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BioRike伯瑞克 > 技术支持 > 技术资料 > SOE-PCR,Fusion-PCR FAQ

SOE-PCR,Fusion-PCR FAQ

作者:admin 发布日期:2018-09-21 18:30
一、
首先从引物着手,分析错配、引物二聚体、发夹环等因素是否有影响。引物没有问题的情况下,按照下面的步
骤进行:
1:两个片段如果是PCR 产物,先回收一下;
2:以这两个片段互为引物和模板,根据片段的浓度可按一定的比例加入;
3:95 度变性3 分钟,(94 度30 秒,72 度2 分钟,25 个循环);
4:电泳检测,理论上应该有3 条带:600bp,1000bp,1600bp;
5:按照目的条带的亮度进行稀释作为模板(有时目的条带根本看不出来,就可以直接作为模板),用两端的
引物再PCR 一次,为提高目的片段的特异性,可把退火温度提高2 度。
 
二、
在实验中,参照Jespeson 等(BioTechniques,23(1),1997, July, p48~52)的方法,为了增加重
叠延伸的概率,我们先建立了不加上下游引物P1a、P4 的反应体系,利用DNA 多聚酶的5'→3'聚合活性及重叠
区的可引发性,在无上、下游引物的情况下以52℃ 为退火温度进行了8 次循环,随后加入引物P1a、P4 在60℃进
行了30 次循环,并获得条带单一的融合基因PCR 产物,从而有效地提高SOE 的效率。另外,为了避免普通Taq 酶
在产物的3'端加上一个模板非依赖性的dATP 而降低SOE 的效率并减少PCR 过程中的错误突变概率,我们选用了
产生钝性末端的Pyrobest 高保真DNA 多聚酶。
 
三、
单基因PCR:以基因1 为模板,以P1、P2 为引物;以基因2 为模板,以P3 和p4 为引物,进行初次PCR 扩
增,分别获得加有重叠引物互补序列的1 和2 基因片段。反应条件:95℃ 预变性5min, 94℃变性1min , 45℃退火
1min, 72℃延伸0.5min, 35 个循环。最后72℃延伸10min . 反应体系为50ul。
重叠延伸反应:将基因1 和2 初次PCR 产物行琼脂糖凝胶电泳回收纯化后,以其为模板、以P1 和P4 为引物
进行重叠延伸反应,获得基因1-2 融合基因片段。反应体系同前,反应条件:95℃预变性5min,94℃变性1min,
50℃退火1min,72℃延伸1min,5 个循环后,72℃延伸10min 终止反应。然后加入引物P1 和P4,将反应条件
改变为:95℃预变性5min,94℃变性1min,45℃退火1min,72℃延伸1min,35 个循环,最后72℃延伸10min。
 
四、
我刚完成一段550BP 和一段1300BP 的连接PCR,方法如下,你可以试一试。
第一轮PCR:
10XPCR buffer 10ul
dNTP          8ul
ddH2O        79.5ul
Ex Taq        0.5ul
fragment1  1ul
fragment2  1ul
94oC 30 s, 68oC 60 s, 20cycles
第二轮PCR:
添加引物各1 ul。
94oC 2 min.
94oC 30 s; 62oC, 30 s; 72oC 1m, 30 cycles
94oC 30 s; 72oC 1 min, 10 cycles

五、
我最近刚好也做了这样的试验,严格说应该叫做重组PCR。前些天测序后已经证明两个片断已经通过18 bp 的
街头连在一起。根据我的经验,我建议楼主这样做:
1.关于四条引物的设计:
a. csa 上游:楼主的引物应该没问题。
b. 下游csa:从5 到3 依次为:街头+CSA 下游。关于街头长度,18bp 已经够用了,楼主用了24 个bp 也
没有问题;CSA 下游引物楼主短了点,17 个bp 也可以,最好在20bp 以上。
c. IFNa 上游: 从5 到3 依次为:街头+IFNa 上游。IFNa 上游引物楼主短了点,最好在20bp 以上。
d. IFNa 下游: 没有问题
2.关于PCR, 我建议楼主这样做:
a. 先分别扩出CSA 和IFN 的目的片断。胶回收。25cycles
b. 重组PCR 楼主可以有两种方法:
1.将回收的两个片断,分别取5-10ul,加上其他PCR 组分至30ul,注意不加任何引物。PCR 10 cycles。然后
取5-10ul 上述PCR 的产物,加PCR 组分至30ul,注意加入csa 上游和IFNa 下游引物。30 cycles.
2. 也可以把第一种方法两个PCR 反应一起做,即直接在第一个PCR 反应中加入引物。30cycles。两个方法我
都能扩出目的片断。
3.关于酶:我用的是KOD plus, toyobo 公司的。楼主的这个酶会有A 加入,建议换。
楼主要先把两个目的片断扩出来。如果楼主两个片断已经分别克隆到载体上,两个片断第二轮PCR 时连不上,
我建议楼主可以把csa 上游引物和 IFN 下游引物分别设计两个,一个用载体的序列,一个用目的片断的,这样先用
载体引物扩出目的片断,回收,然后再用片断引物作重组PCR,有点类似套式PCR。
 
六、
http://www.archimedes.rwth-aachen.de/MolbioProtocols/Phage%20Display/PCR%20amplification%20of
%20variable%20regions%20(SOE-Protocol).html
The SOE-Protocol given below utilises our initial set of primers for mouse and chiocken phage display
wherby the amplified VH and VL are connected by a primer encoded (Gly4Ser)3-linker.
For the amplification of the variable regions, three different murine primer sets can be used:
VH front (MuPD 3, 4, 5, 6, 7. 8. 9. 10, 11 and 12, mix) and VH back (MuPD 34, 35, 36 and 37, mix) for VH
amplification, CPDVHF and CPD VH Gly for chicken VH.
VK front (MuPD 19, 20, 21, 22, and 23, mix) and VK back (MuPD 27, 28 and 29, mix) for VK amplification,
CPDVLF and CPD VL Ser for chicken VL.
Vlambda front (MuPD 26) and Vlambda back (MuPD 30) for Vlambda amplification.
Every PCR amplification of variable regions was done using 4 to 10 μl of the first strand reaction.
Set up the following PCR reactions (50 μl total volume each, combine 10 pM of forward primer with a total
of 10 to max 20 pM of a mixture of all backward primers):
VH amplification from G1 first strand
5-10 μl first-strand reaction (with COH 30 primer)
2 μl VH front primer (10 pmol/μl stock)
2 μl VH back primer mix (10 -20 pmol/μl stock)
2,5 μl DMSO
4 to 4.5 μl PCR 10x buffer
3 μl MgCl2, 25 mM
0,3 μl Taq-polymerase (5U/μl)
ad 50 μl Water
VH amplification from G2a/2b first strand
5-10 μl first-strand reaction (with COH 32 primer)
2 μl VH front primer (10 pmol/μl stock)
2 μl VH back primer mix (10 -20 pmol/μl stock)
2,5 μl DMSO
4 to 4.5 μl PCR 10x buffer
3 μl MgCl2, 25 mM
37
0,3 μl Taq-polymerase (5U/μl)
ad 50 μl Water
Vkappa amplification from kappa first strand
5-10 μl first-strand reaction (with MuPD 31 primer)
2 μl VH front primer (10 pmol/μl stock)
2 μl VH back primer mix (10 -20 pmol/μl stock)
2,5 μl DMSO
4 to 4.5 μl PCR 10x buffer
3 μl MgCl2, 25 mM
0,3 μl Taq-polymerase (5U/μl)
ad 50 μl Water
Vlambda amplification from lambda first strand
5-10 μl first-strand reaction (with MuPD 31 primer)
2 μl VH front primer (10 pmol/μl stock)
2 μl VH back primer mix (10 -20 pmol/μl stock)
2,5 μl DMSO
4 to 4.5 μl PCR 10x buffer
3 μl MgCl2, 25 mM
0,3 μl Taq-polymerase (5U/μl)
ad 50 μl Water
Start the following program:
5 min 95°C
then 30 times:
40s 95°C
2 min 58°C
2 min 72°C
final:
5 min 72°C
Check reactions putting 5 μl on a 1,2 % agarose gel.
Purify amplified variable regions by agarose gel (1,2 %) electrophoresis and Gelclean (Macherey Nagel):
Load each PCR reaction in one slot of 40 μl, store the remaining 5 μl PCR reaction at -20°C. Later, this can
be used for reamplification, if necessary. Cut out the right bands and purify. Two elutions can be done with
30 μl Tris/EDTA buffer, the two elutions are combined (±55 μl final volume per PCR reaction). Check
purifications, putting 3 μl of each purification on a 1,2% agarose gel. Calculate the concentration of each
purified PCR product.
Making scFv's by SOE-PCR
In the SOE-PCR the VH and VL regions are combined randomly by their overlapping linker region. We
choose to mix equal amounts of variable regions from heavy chain (G1, G2a/2b, mix 50:50), and light chain
(K and lambda regions, mix 95:5). In the 'second step PCR' the synthesized scFv encoding sequences from
the SOE-PCR are amplified using two primer mixes: univ VH (MuPD 18) and univ VL (MuPD 33). Finally,
these scFv sequences contain SfiI and NotI restriction sites at their borders, so they can be cloned in the
phage display vector pHEN1. It is very!!! important to combine equal amounts of variable heavy and light
regions for the SOE-PCR. We had good results using 12,5 ng of each variable region, that means 50 ng
variable regions in total per SOE. The amount of SOE-PCRs that can be done with the purified variable
regions is limited by the variable region with the lowest purification yield. Using more variable regions
than 12,5 ng can give more scFv product, but then you can do less SOE-PCRs. Each SOE-PCR can be
divided in five second step PCRs.
For each SOE-PCR, mix the following components:
x μl VH G1
x μl VH G2
x μl VK
x μl Vlambda
3 μl MgCl2
5 μl PCR buffer
4 μl 2,5 mM dNTP
0,2 μl Taq polymerase
x μl H20
50 μl Total volume
Add 50 μl mineral oil and run the following program in the thermocycler (7 cycles):
5 min 95°C
2 min 55°C
10 min 72°C
For the second step PCR, mix the following components:
10 μl SOE-PCR
4 μl PCR buffer
2,4 μl MgCl2
4 μl 2,5 mM dNTP
0,25 μl univ VH primer (10 pM)
0,25 μl univ VL primer (10 pM)
0,2 μl Taq polymerase
28,9 μl H20
50 μl Total volume
Note: using 10 pmole of each primer instead of 2,5 pmole gave an additional PCR product at ±400 bp!!!
Add 50 μl mineral oil and run the following program in the thermocycler:
15 min 95°C
then 30 times:
2 min 60°C
3 min 72°C
1 min 94°C
final:
2 min 60°C
10 min 72°C
Check the PCR reaction by running 5 μl PCR reaction on a 0,8% agarose gel.
Purification of the scFv fragments
The scFv fragments are purified from the PCR reaction by special gel filtration, using S-400 sephacryl
columns from PHARMACIA (Cat. No. 27-5140-01). These columns take out primers and other PCR material.
It is much faster than gel purification. Besides, gel purification can give bad ligation efficiency.
Combine all second step PCR reactions and add 1 μl protease K to release any Taq, sticking to the DNA
and incubate for 30 min at 50°C
Add 1 vol PCI, vortex shortly, centrifuge 5 min in microcentrifuge, take off superna-tant and purify the scfv
fragments in the supernatant through a S-400 sephacryl micros-pin column:
a. vortex the micro-spin column to homogenize
b. screw off the top cap but leave it on
c. break off the bottom cap
d. put the column in a eppendorf tube and spin for 1 min at 735 xg
e. pipet the supernatant on the top of the resin, do not touch the resin.
f. spin 2 min at 735 xg, remove the column
g. spin the eluate 5 min at topspeed to remove remaining resin
h. Pipet the supernatant in a new eppendorf
Note: Do one column purification for each two second step PCRs (a. 100 μl).
Before doing the restriction digest, the purified scFv was concentrated by precipitation:
a. Pool scFv purifications
b. Add 1/10 volume 2M NaCl and 3 volumes ice cold ethanol
c. Incubate overnight at -20°C
d. Centrifuge 10 min topspeed in a microcentrifuge at 4°C
e. Take off supernatant
f. Wash the pellet with 200 μl 70% ethanol (roomtemp)
g. Spin 5 min at roomtemp, topspeed
h. Take off supernatant
i. Dissolve the pellet in 10 μl H20.

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