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PCR not working

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11 replies to this topic

#1 molbio1234

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Posted 09 April 2014 - 11:51 AM

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.



#2 jerryshelly1

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Posted 09 April 2014 - 12:01 PM

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?



#3 molbio1234

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Posted 09 April 2014 - 12:08 PM

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


Edited by molbio1234, 09 April 2014 - 01:07 PM.


#4 jerryshelly1

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Posted 09 April 2014 - 12:25 PM

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.



#5 molbio1234

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Posted 09 April 2014 - 12:29 PM

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.



#6 hobglobin

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Posted 09 April 2014 - 12:37 PM

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?


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#7 molbio1234

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Posted 09 April 2014 - 01:08 PM

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.



#8 jerryshelly1

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Posted 09 April 2014 - 01:30 PM

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.



#9 Trof

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Posted 09 April 2014 - 03:42 PM

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?)


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#10 molbio1234

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Posted 09 April 2014 - 03:59 PM

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.


Edited by molbio1234, 09 April 2014 - 04:02 PM.


#11 Trof

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Posted 10 April 2014 - 04:07 AM

I use this tool, EMBOSS Cpgplot but usualy decrease windows size to around 20 to get higher resolution. It's not a simple GC content, but a graph of relative GC % over a set region. Amplicon may have average GC but at the same time have a high GC region, that makes problems.


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#12 jerryshelly1

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Posted 10 April 2014 - 10:18 AM

You can't run them together. I just open two windows and compare them. If you are looking to find a common Tm, try NEB's Tm calculator.

 

You could always consult Emsembl or Genome Browser to see relative GC content across your gene of interest.







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