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problem in pcr and purification


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3 replies to this topic

#1 anuj

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Posted 25 September 2009 - 05:52 AM

i am having a template of 387bp. i have 3 set of reverse and forward primers but with different restriction sites.
on small scale all pcr works good at annealing temp of 58. but at time of large scale with same set of primer sometime one is not amplifying , 2nd time some other is not amplified under same set of conditions.
don't know why is it happening?

also after purification i get very diluted(faint) product. i've tried purification with both gel and kit method. i use RBC kit.
suggest some other method of purification so that i can have concentrated purified pcr product.
plz suggest some solution.

#2 Adrian K

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Posted 06 October 2009 - 07:22 PM

sometime is like that...try a gradient on your "large scale"...

suppose you use HiYield™ Gel/PCR DNA Extraction Kit .
final elution time use your lowest volume (20ul)....make sure you put in all yr pcr products in gel.
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#3 swanny

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Posted 06 October 2009 - 09:54 PM

i am having a template of 387bp. i have 3 set of reverse and forward primers but with different restriction sites.
on small scale all pcr works good at annealing temp of 58. but at time of large scale with same set of primer sometime one is not amplifying , 2nd time some other is not amplified under same set of conditions.
don't know why is it happening?

also after purification i get very diluted(faint) product. i've tried purification with both gel and kit method. i use RBC kit.
suggest some other method of purification so that i can have concentrated purified pcr product.
plz suggest some solution.

What's the difference between the 'small scale' and 'large scale' reactions? What are your temperatures and reaction times? If you increase the reaction volume by more than, say, 2x, the centre of your tubes might not reach temperature.

As an alternate to gradient PCR, try a touchdown PCR.
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#4 perneseblue

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Posted 07 October 2009 - 03:50 AM

What's the difference between the 'small scale' and 'large scale' reactions? What are your temperatures and reaction times? If you increase the reaction volume by more than, say, 2x, the centre of your tubes might not reach temperature.


To add to swanny's point, it is sometimes easier to simply do several "small scale" reactions than it is to do one "large scale reaction", for the purpose of obtaining sufficient material.
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