Dear everyone
I tried to amplify my PCR product which has a size of 1800 bases by using Phusion DNA polymerase enz. for many times.
Even I followed the PCR reaction and PCR condition according to manufacturer recommendation but I also did not get the correct product size.
I also tried to use the GC buffer and varied the concentration of DMSO, changed the primer design but I never get the PCR product, again.
Could anyone suggest any other ideas that I missing?
Thank you very much
Did you check the template?
Do you get some other band so is your enzyme active?
assuming you might have tried multiple ways and yet no amplification, still more information like the amount of template used, presence of primer dimer in gel after agrose run, etc is needed to help you out
Hi there,
How did you get this PCR product at first? If you know the protocol you should be able to reproduce/adapt it. Otherwise there is something wrong with the template, the primers, the PCR recipe, the Phusion... So first I would suggest to run a control PCR with primers and Template which you know they work OK. Once this down it would tell you there is something wrong specific to your Template/primers system.
Check the sequence of the amplicon, if it is high GC content you will likely have trouble but increasing your concentration up to 8% DMSO may do the trick. Also if you have a gradient thermocycler, try running the PCR using annealing temperature gradient between 55-70C, 35 cycles. Do this with and without DMSO using GC Buffer.
What is the template, plasmid, gDNA, or cDNA? Make sure it is intact by running on gel. Don't use too much.
Also ensure that the enzyme is working by performing another control PCR ideally with the same template to rule out those issues, as others have said.
If your control works, and you still can't get the PCR with Phusion, try PrimeStar Max or CloneAmp HiFi from Takara.
our experience with Phusion is that reaction volumes should mostly also be considered, even if you don't expect
in some cases small volumes work well in others larger
Can you tell us more about the reaction? Is this routine amplification? Cloning? Mutagenesis? What is the source of your DNA template? If you're engineering some function in your primers (mismatches, for example), the polymerase can chew up your amplification product after too many cycles. Like others have said before, the control reaction would be super helpful here.
Hi Kwanhathai Sinsirimongkol,
What I understand from your explanations; you might be trying to amplify your target gene (1.8 Kb) from cDNA library for cloning purpose using cloning-primers.
If I am right, then I can suggest some points:
1- Check the abundance of your target gene by qRT PCR in your cDNA. Then fine tune the cycle no. for your full length gene amplification.
2- You can prepare your own cDNA (library) by gene specific single Rv. primer instead of Oligo-dT.
3- Follow primer blast to design primer and to avoid non-specific binding to other cDNAs. However, if you follow above Point-2 you have less probability for non-specificity.
4- Since you are designing cloning primer, keep the length of the primer from 25-30 bases max. And min. 18 bases for complementary binding for the first cycle. Tm should be around 70C and min 10C more than the annealing temperature.
5- If cloning-primer non specificity is a problem then you can design primers from 5'UTR and 3'-UTR regions close to the CDS.
6- Put a Temperature gradient for better results (as suggested before).
7- Sometime at constant (or low) KCl with more (gradient) MgCl2 gives better results for longer products. Please read the following link...
https://www.biotecharticles.com/Biotech-Research-Article/Role-of-KCl-and-MgCl2-in-PCR-3271.html
8- To multiply your target gene better you can use your 1st PCR amplicons as template for a 2nd PCR reaction.
I think this may help you as well as others.
Good luck.
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