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PCR troubleshoot

pcr

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#1 kidakarn

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Posted 31 August 2014 - 05:37 PM

Hello, Im a graduate student. Now, I working on part of PCR lab. The problem is I use the condition that my adviser was use and it give a good result with a thick band of PCR product but last two week I perform the experiment with the same protocol as previous and I got the faint band. So help me please.



#2 bob1

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Posted 31 August 2014 - 08:45 PM

Most likely one of the components has gone off, probably the dNTPs. Make fresh aliquots of the dNTPs, the primers and your template DNA and test them.



#3 kidakarn

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Posted 05 September 2014 - 05:15 PM

Thank you a lot,

 

However, now I use new master mix and also order a new set of primer.

When I use 25 ul/reaction and load 5 ul into gel it give a good band but when I make 100 ul/reaction band is faint than 25 ul/reaction.

help me please.



#4 bob1

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Posted 06 September 2014 - 11:03 AM

So are the concentrations of reagents different between the two volumes? How much DNA do you add to each? Are the cycling conditions the same? Are you mixing properly?



#5 mdfenko

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Posted 08 September 2014 - 03:46 AM

keep in mind that the thermal characteristics of 100ul are different from those of 25ul. you may want to try making 4 X 25ul reactions and combining them after cycling.


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#6 Ameya P

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Posted 09 October 2014 - 11:55 PM

keep in mind that the thermal characteristics of 100ul are different from those of 25ul. you may want to try making 4 X 25ul reactions and combining them after cycling.

 

Yes, this what I would do, if I wanted more product.

 

Recently, when I sent a sequencing provider multiple tubes of the sample sample , so that they could be used in case additional sample was required, he reverted back saying that " PCR IN EACH TUBE WORKS DIFFERENTLY and products from different tubes should not be mixed". ohmy.png


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