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PCR not working - (Apr/09/2014 )

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I am not getting PCR products of a particular gene called ABCD1 using normal PCR of denature, anneal, and extension, or when using a touchdown PCR on the same gene. I quantified my DNA sample and primers. The primers have GC content in range from lowest at 50C and highest at 77C. Is the GC content of the primers too high? Any suggestions on what I could try or look into? Has anyone amplified this gene before? Would appreciate some insight.

-molbio1234-

It sounds like the annealing temperatures of your primers are way to far off. Does the sequence of ABCD1 make it impossible to design primers that have similiar annealing temps? Are you able to post your primer sequences?

-jerryshelly1-

the Tm ranged from 52C to 64C. the annealing temp on the normal PCR was the median of this range, or 58C.

-molbio1234-

What exactly are you trying to do? Are you setting up a standard PCR? You could trying running a gradient PCR or add betaine or DMSO to your reactions.

-jerryshelly1-

I am simply trying to amplify the template for the exons of the gene. I have done a touchdown with annealing temp from 65C, to 55C, but that didn't work. What is the gradient PCR setup? I never did that.

-molbio1234-

A gradient PCR uses a gradient of possible annealing temps within the range of predicted ones... in your case perhaps 50 to 60/65°C. Then you find hopefully a temperature with a working and specific reaction.

What about cycling conditions and the reaction mix?

-hobglobin-

the cycling conditions are standard, I am doing 30 cycles total. the reaction mix is standard per the taq I am using. I am not adding in betaine or DMSO or MgCl2. is this crucial given the ranges of Tm and GC % I posted? the protocol that came with the taq I use does not say that these are required, only additional additives.

-molbio1234-

Silly question, but you are trying to amplify each exon independently correct... Running your FWD/REV primer pairs through NEB and IDT gives a two Tm's that are relatively close. Make sure that your primers and all of your reagents are diluted properly before running and give gradient PCR a try.

-jerryshelly1-

What is the GC content of the amplicon? If it contains regions with high GC, then you should not discard the DMSO/betaine so quickly. These are usually not needed when amplicon GC is normal, but are required if there are GC rich regions. Taq can't proceede through such places and stalls amplification. GC of primes is not that important (though it should be between 20-80).

If you are sure you have primer design right, tried all temperatures and a touchdown protocol, then try adding 5% DMSO to it.

 

(just.. not adding MgCl2 is fine if you aleady have enough in your buffer/have an optimized amount etc.. do you?)

-Trof-

Silly question, but you are trying to amplify each exon independently correct... Running your FWD/REV primer pairs through NEB and IDT gives a two Tm's that are relatively close. Make sure that your primers and all of your reagents are diluted properly before running and give gradient PCR a try.

 

jerry, can you post the link to IDT website? I have ordered the primers through IDT but I don't know where I can run my F and R primers together on the website.

 

Trof, I don't know the GC content of each amplicon (exon). Is there a website that allows me to search on a gene and its exons, and displays GC content of that exon and neighboring regions? that will be helpful to know.

-molbio1234-
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