q*****d
发帖数: 445 | 1 最近要做library, 使用这个实验室的方法进行易错PCR
（http://www.msg.ucsf.edu/agard/Protocols/PCR_Random_Mutagenesis.htm），酶
是NEB的Taq,但是效果一直不是很好，35 cycle PCR的浓度还是很低，30cycle特别的少
，请问
大家有什么方法提高产量吗？还是我的方法不够好，谢谢大家。
PCR Random Mutagenesis
Materials
1. Parental plasmid
2. Oligos surrounding region to be mutagenized
3. Error-prone PCR buffer (10x is 100mM Tris-HCl, pH8.3; 500mM KCl,
70mM MgCl2, 0.1% (w/v) gelatin.)
4. 10mM MnCl2 (stock) (make sure there is no brown coloring to
stock soln; if there is it means it oxidized to Mn3+ which will kill
your polymerase)
5. dNTPs
6. Taq polymerase
Method
Add: 10l of 10X Error-prone PCR buffer,
10l DMSO (for GC rich template regions)
3l of 10mM MnCl2
50pmol of each primer,
10ng of template plasmid,
dNTPs
to 0.2mM of dATP and dGTP;
to 1mM of dCTP and dTTP
5U of Taq polymerase
H2O to 100l
-It is preferrable to then split this stock into 10l
aliquots and then run these reactions separate and then mix them
together after completion of the reaction.
PCR: 94° for 30s
55° for 1min
72° for 1min
repeat for 30 cycles
Notes:
1) The PCR cycle temps and times listed above are for PCRing aLP DNA
and will probably need to be changed to suit the needs of you and your
template.
2) The MnCl2 concentration can be changed to alter the mutagenesis
rate according to your wants and needs as an experimental research
scientist. The above protocol calls for 0.3mM MnCl2 which will give an
approximate 5 point mutations/kb.
3) The separation of PCR master mix into different aliquots prevents
the over-representation of single mutations in your library. This is
because if a mutation occurs in the first rounds of PCR, then this
mutation will be present in most or all of the subsequent products,
thereby reducing the diversity of your library.
4) Even though PCR mutagenesis is rather robust, there still is an
inherent mutagenic bias associated with this method. There is a PCR
mutagenesis kit sold by Stratagene (the GeneMorph system using Mutazyme
polymerase) that has an opposite bias. A combination of these two
methods should produce the most diverse library. (For a very good
representation do 15 rounds of this PCR followed by 15 rounds of
Mutazyme.) | H****s
发帖数: 301 | 2 GeneMorph II Random Mutagenesis Kit | q*****d
发帖数: 445 | 3 谢谢回复，要是用这个，就不用发贴请教了！
【在 H****s 的大作中提到】
: GeneMorph II Random Mutagenesis Kit
| w**t
发帖数: 52 | 4 我以前也遇到这种问题，你可以尝试使用dITP，不过有一定的bias，或者是用chemical
mutagensis。
不过最简单应该是多做几管PCR吧，反正taq便宜啊。 | s********n
发帖数: 2939 | | a***e
发帖数: 1010 | 6 computer mutagenize, then chemical synthesize. | M*****n
发帖数: 16729 | 7 why not 40 cycles? we routinely do 40 cycles | q*****d
发帖数: 445 | 8 谢谢，我再增加cycle试一试！
【在 M*****n 的大作中提到】
: why not 40 cycles? we routinely do 40 cycles
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