Hi
I am trying to amplify a 3 kb gene from Mtb genomic DNA using phusion polymerase but we are not getting any amplification. We are using GC buffer. We are using 100ng/ul DNA, we have tried gradient of 55-70 deg and extension time of 1 min 30 sec. We have also tried PCR with different additives like 0.75% DMSO final concentration and 5% 2-pyrrolidinone.
Even Q5 reaction mix didnot work. Please suggest.
I think the extension time is too short, try 5 minute extension time reducing it to 3 mins if it works at 5mins
Hi there,
Did you check that your DNA is efficiently used as a template for a non problematic amplification? I'm wondering about the quantity of DNA in your reaction? DMSO may also be tried up to much higher percentage (I usually do 3% for difficult cases).
Trouble-shoot this step by step. First, can you amplify a smaller product from these DNA samples (like just CO1)? Do you have a good positive control DNA sample?
Second, increase the extension time to at least 3 minutes.
What temperature was recommended with your primers? Try the gradient with the longer extension time AFTER you have verified that you can amplify a shorter PCR product from your DNA samples.
Also, I think you are using WAY too much DNA. Less often works better.
Good luck!
Hi Paul
I kept the extension time short because it was recommended in protocol of fusion pol.
@Dominique: I checked lot of genomic DNA for this purpose. I even made a fresh g. dna for this amplification. I can increase DMSO and will also put a ctrl pcr for checking amplification.
@Katie: thanks Katie.. I would try a ctrl reaction as well and I used 1 min 30 sec as fusion pol processes 1kb/30 sec as in the manual. I can try increasing extension and decreasing g dna conc.
Sometimes to amplify GC rich region from Mtb genome is little tricky. I had a issue in the past( to amplify Rv0574c ) but I did some correction in term of primer Tm during the time of synthesis and DMSO concentration in PCR reaction. The Tm should not be more than 65 (check before sending for synthesis) and you can use DMSO concentration till 3%.
I hope this will work.
Good luck
@ Shashi sir: Sir I remember Rv0574c, we used to add DMSO and it used to work but this is three times length of Rv057c. As per idt Tm calculator the tm of one primer is 60 and other one is 70. After seeing the suggestions I am trying a higher extension time as well as DMSO concentration in a gradient from 55 -70. please tell me whether its fine?
3 kb amplification is fine. You should make primer again. This primer will not work because the difference of Tm is 10 degree. If Tm for both primers is 70 degree then u should add DMSO to decrease the Tm equally for both the primers.
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