DMSO in PCR

1. 如果要增加特异性，要考虑是否升温，复性时候的较高温度有利于筛选特定片段，减少其他GC丰富片段的竞争。

2. 考虑使用甘油：加甘油也可以的,一般终浓度在10%左右.我的实验也曾遇到过GC高达73%的情况,加过甘油非特异性改善了很多,你不妨试一下.

3. 使用甜菜碱：

DMSO can usually give results in GC-rich regions when used at a final

concentration of 5%, although there are reports in the literature of

genes which required 10% DMSO, so a bit of experimentation may be

necessary to optimise the concentration for your genes. Alternatively,

addition of Betaine to concentrations of 1M in reactions can also be

beneficial in GC-rich regions.

There was a comparison published in Promega Notes 65 p 27-29 where the effectiveness of betaine and DMSO, used separately and together in

reactions, where compared to commercially available PCR additives, along with NMR spectra confirming that some of these expensive additives are

little more than betaine! In this study, the use of 5% DMSO alone

produced a number of non-specific bands, and a strong specific band,

while 1M betaine used alone resulted in the specific band alone, but

probably about half the amount of product. Combining DMSO and betaine together in the reaction resulted in the single specific band, but

perhaps in slightly greater proportions than when using betaine alone.

甘油ji及DMSO等PCR佐剂是增加特异性的,前提是你的产物中有杂带, 但是目的条带也有的情况下才加, 因为PCR佐剂在增加特异性的同时也会降低PCR的产量即PCR的效率.

而且,这2种不必同时加,加5%的甘油或者10%的DMSO即可.

4. DMSO反应体系中未加入DMSO时出现多条低效扩增带 , 经测序证实全为非特异扩增带. 如加入 DMSO 后仅有一条高效扩增带 , 测序证实正是目标片段 (图略) . 另一方面 ,DMSO浓度过高时又同时使 Taq酶变性失活 , 反而降低扩增效率. (附图 b) 中DMSO浓度从 14%～20%的过程就是 Taq酶活性逐渐被抑制直至几乎完全丧失的过程. 有学者认为 ,PCR 反应中 DMSO 的加入量以 019%～2%为最佳 , 如达 10%则 Taq 酶活性会被明显抑制.这可能是针对 50μL 反应体系含 215UTaq 酶的标准反应而言. 我们的结果表明 , 为得到最佳扩增效果 , 应使用较高浓度的 DMSO, 由此造成的对酶活性的影响可经增加 Taq酶用量 (5U/50μL反应体系) 来消除. 这样虽酶的用量加了倍 , 但因扩增效率和成功率的大大提高而避免了更多的浪费.

5. 尝试使用BSA Various authors recommend DMSO and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5-10% (v/v). In the multiplex reaction, however, these adjuvants gave conflicting results. For example, 5% DMSO improved the amplification of some products, decreased the amount of others whereas some loci were not influenced at all. Similar results were obtained with 5% glycerol .

BSA, in concentrations of up to 0.8µg/µl appeared to increase the efficiency of the

PCR reaction much more than either DMSO or glycerol. It should be noted that BSA did not have an inhibitory effect on any of the loci amplified.

6. Bovine Serum Albumin (BSA):

100 μg/ml - 1 ug/ul, 0.01% - 0.1% (w/v)

The addition of albumin to tissue DNA samples increases theamount of DNA generated by neutralizing many deleterious factorsfound in tissue samples which can inhibit PCR. Concentrations ofup to 0.8 μg/μL may increase the efficiency of a PCR reaction much more than either DMSO or glycerol.

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