Hello, everyone
I have been struggling to amplify a mouse promotor for almost 2 months now. This fragment is:
- Roughly 1500-1700 pb depending on the set of primers
- GC content of about 60%, with several regions over 75%
At the beginning I did not even get bands at all. Recently, when I bought a new forward primer, I at least got bands of 700-800 pb and under. However, for a while now, no matter what I change:
- Annealing temperature, which I went to as down as 48ºC
- Extension time 1:30-2 minutes
- initial denaturalization 5 minutes at 95ºC
- DMSO 0-1-2-3-4-5%
- Betaine 0.5M-1M-1.5M-2M-2.5M
- DMSO and Betaine together (5%DMSO and 1M Betaine)
I always get the same 800 pb size band. Does anyone have an idea? This is the first primer that at least have amplified something bigger, the maximum I got before this primer was 300 pb.
Thanks in advance
Hi Beatriz,
Perhaps you may have better luck if you used a different polymerase.
NEB has a number of polymerases and each could yield different specificity and thus multiple bands or no band depending on the choice of primers and templates.
Phusion is high fidelity while OneTaq is for routine PCR.
I have found that sometimes using these to polymerases I get different results.
Both come with GC buffer as well which may provide additional options.
For annealing tempterature I use Tm calculator, which is an online software by NEB.
I have noticed that different polymerases have different annealing temperatures with the same primer-template pair.
Ideally when you design primers their Tm should not differ by more than 3 degrees, which you may evaluate and adjust using Tm calculator.
Hope this helps,
Best,
Sudeep
One more suggestion.
When you design primers also look for secondary targets in your template.
Such as if you are using mouse genomic DNA then use the mouse genomic DNA as template while doing the primer blast using NCBI software.
In case you have several secondary targets, I will suggest you to do two rounds of PCR. One with a longer template which provides unique primers and that encompasses your target sequence.
So after the first round of PCR, gel purify the PCR product and then amplify your target DNA with the second set of primers using the PCR product as template.
Thereby eliminating most of the secondary targets, which cause interference in your amplification.
best,
Sudeep
You have tried some excellent remedies and the answer of Sudeep Kumar is a very good idea. One other good try is the use of 7 deaza dGTP to replace about 1/4 of the dGTP as in the attached paper.This minimises triple hydrogen bonding and works very well. The authors also suggest using dITP as used in sequencing chemistry kits to get through high GC regions but many polymerases fail to incorporate the I analogue properly above 60c so this is less useful. Other tries are touchdown pcr and increasing the chance of longmers amplifying by increasing the extension time of your pcr but I suspect that your short product is caused by looping in your target dna producing hairpins so keep the dmso and betaine.Use a hot start enzyme and some 7deaza dGTP and rue your bad luck in choosing such a high GC amplimer. If absolutely everything fails consider 2 shorter overlapping amplimers to be joined later if required by melting and re annealing. Clearly your pcr primers should anneal at the highest possible temperature to stand any chance of annealing and extending before the template re anneals
the 800 band is probably a short amplification caused by the high GC region forming a loop and the enzyme reading across the loop so not amplifying the sequence within the loop
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