no bands in my PCR - (Jul/23/2009 )

What to do if I have no bands in my PCR?
I use 4 mM MgCl2, 2x as much DNA as usual, 2x as much Taq as usual, I run 40 cycles @ 55 degrees celcius. What else can I try?

Ytje

Ytje on Jul 23 2009, 05:34 AM said:

Many things can be interrogated and refined.

1. Redesign primers.

2. Make sure your DNA template is good (not too fragmented), pure (no phenol, proteinaseK), and has the right amount.

3. If it is a big product you are looking for, you need special Taqs/mixes.

4. Try various concentrations of MgCl2, and do a gradient PCR to find the optimum annealing temp.

5. Do a hot-start.

5. If it is GC rich region, adding DMSO may help.

Ytje on Jul 23 2009, 09:34 PM said:

try gradient PCR first before you do anything else. If you can provide us more information maybe we can help.

Also, monitor your extension and annealing time.

Hope this helps. Let us know the outcome.

Thanks for your help!

We don't have a PCR machine that can do a gradient PCR. We tried 55, 58, 60 and 62 deg in different blocks.

I now tried using plasmid DNA to get at least some results.
I got my signal at 55 deg, different MgCl2 concentrations and by adding DMSO or BSA. I chose 55 deg because it seems to me that the lower the temp the best chance you have...
So now I'm going back to the DNA from mouse tails to see if that works. The problem could very well be that the DNA is not pure enough (primers are not the problem, I guess).

Ytje

Hi, Ytje

IS good to hear that you got your product at 55C. If your PCR machine can do touchdown, it will be good to try on annealing temperature from 57 to 54 (-0.2c per cycle) for first 15 cycles, and for the last 15 cycles try, run 54 C annealing temperature. Hope this might help.

Adrian

Hi Adrian,

Isn't touchdown meant to get rid of non-specific amplification?
Why do you think this will help?

Sorry, I don't mean to be rude. I'm just curious.

Ytje

Hi Ytje,
yes, touchdown can and usually is use to get rid of unspecific band.

For my past experience my pcr condition was working on plasmid, however when I use genomic dna, it appears some unspecific band, even I had tried out gradient. I solve it by using touchdown pcr.

So lets say that If you use your mouse genome and get unspecific bands (touchwood, just in case, no offence...), and you unable to do gradient, touchdown might hopefully helps you.

Just my 2 cents experience to share.
Adrian

Thanks Adrian, I'll give it a shot

Ytje

Adrian,

I found a PCR machine that can do gradient PCR!
What gradient do you suggest I should do? or is it a fixed protocol?

Ytje

Ytje,

Hi, the gradient temperature is to find the optimum annealing temperature for your PCR. in order to determine which is your annealing temperature, you had to find out whether your primer was used by any publications before. If yes, do a gradient range + and - 4C from the published temperature, eg: if the published temperature is 55, try a range from 51 to 59 to see whether the published temperature is the optimum temperature for you. The variation happen is due to different PCR machine, different PCR tube heat transfer and maybe some of the thermalcycler internal settings. If the your primer is self-design, try this formula: Tm = 4°C x (number of G’s and C’s in the primer) + 2°C x (number of A’s and T’s in the primer), then do a range of + and - 4C from the calculated temperature.

For the extension time (72 C) . it should be note that 1 minute required per 1kb gene amplification.

Since you had mentioned that you got your signal at 55C, with various MgCl2 DMSO /BSA. Try the same mixture recipe and do a range of gradient temperature from 51 to 59, or from 53 to 59 (or any range you feel suitable) to determine the strongest signal.

Let us know your outcome and I wish you all the best in your work. May god bless you!

Adrian
p/s: I'm quite busy these days doing my work and seldom online....sorry for the late reply.