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.
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6 replies to this topic
#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". 
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#7
kidakarn
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Posted 26 March 2015 - 07:03 AM
Thanks a lot everyone, it has been a long time that Im not log in this site.
Now, Im already solved this problem. 
And tmr I will have thesis defense.
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