6 replies to this topic

[OA17]

Hi!

I have been encountering some difficulties for reamplifying some PCR products. I have heard about using DMSO or BSA to enhance the polymerase activity, but I have never tried.

Can somebody tell me what is the exact function of DMSO or BSA, when to use each one, and at which concentration should I use them? Does it work with any polymerase?

Is it true that for some polymerases, adding the PCR reaction buffer at 2X instead of 1X is good for having more product?

Thank you!

[lab rat]

DMSO and BSA are adjuvants that make the DNA more accessible to the enzyme for amplification. DMSO improves strand separation in G-C rich regions. BSA also improves specificity in amplification of regions with secondary structures. Betaine and glycerol can also be used.

The concentration depends on the template, but as a general rule, use 5% DMSO or 0.5 ug/ul BSA (final concentration). Perhaps someone else can verify that these adjuvants work with any polymerase?

I have accidentally used too much 10X buffer, and my reactions didn't work. I can't vouch for an increase in template amplification.

[KimWG]

lab rat, on Dec 8 2009, 09:52 AM, said:

DMSO and BSA are adjuvants that make the DNA more accessible to the enzyme for amplification. DMSO improves strand separation in G-C rich regions. BSA also improves specificity in amplification of regions with secondary structures. Betaine and glycerol can also be used.

The concentration depends on the template, but as a general rule, use 5% DMSO or 0.5 ug/ul BSA (final concentration). Perhaps someone else can verify that these adjuvants work with any polymerase?

I have accidentally used too much 10X buffer, and my reactions didn't work. I can't vouch for an increase in template amplification.

Hi,

I am by no means an expert -- I started reading your thread because I have my own PCR problems. But I have had BSA help me a lot with amplifying from some extractions that had previously failed. From what I have read, BSA is particularly useful when there are PCR inhibitors in the extraction. I use it at the concentration suggested above, have tried it with three different kinds of taq, without problems. I am working with fecal samples with lots of degraded DNA and PCR inhibitors, and BSA has been the most useful addition to my PCR's, hands down.

I have not tried DMSO, but from what I have read it is more useful for reducing non-specific products and does reduce Taq activity. Betaine is also suggested as an alternative for GC-rich regions, which does not reduce Taq activity, I have again used this without problem. I do not recall the final concentration, but a google search should turn it up. Qiagen's special "Q-solution" is supposedly just 5M betaine. If you are not getting enough product, you may want to try increasing the Mg concentration up to 3 or 4 mM final concentration. This supposedly strengthens all hydrogen bonds, and would increase both the amount of target and nonspecific product produced.

Kim

[Adrian K]

OA17, on Dec 8 2009, 08:36 PM, said:

Hi!

I have been encountering some difficulties for reamplifying some PCR products. I have heard about using DMSO or BSA to enhance the polymerase activity, but I have never tried.

Can somebody tell me what is the exact function of DMSO or BSA, when to use each one, and at which concentration should I use them? Does it work with any polymerase?

Is it true that for some polymerases, adding the PCR reaction buffer at 2X instead of 1X is good for having more product?

Thank you!

Lab Rat and KimWG had explained it all regarding DMSO and BSA.
I would just like to share my experience, I use DMSO in my PCR even in my multiplex PCR. 2X PCR reaction buffer did not yield more product for me in my case, and it does inhibit my PCR reaction (PCR buffer less than 0.8X or more than 1.5X doesn't work for me.).

[lab rat]

Quote

Lab Rat and KimWG had explained it all regarding DMSO and BSA.
I would just like to share my experience, I use DMSO in my PCR even in my multiplex PCR. 2X PCR reaction buffer did not yield more product for me in my case, and it does inhibit my PCR reaction (PCR buffer less than 0.8X or more than 1.5X doesn't work for me.).

I had never heard of using DMSO in multiplex PCR. (I learned something new today.) Does it have a significant effect?

[Adrian K]

Hi lab rat, for my experience without it there are some smears and some bands become more faint...

[OA17]

Ok. I will try BSA and DMSO at first.
Thank you very much for your helpful explanations!