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All gradient samples give bands! How come?


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4 replies to this topic

#1 Curtis

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Excellent

Posted 11 July 2009 - 07:13 AM

I just received new primers so i ran a gradient PCR to determine best annealing temp.

surprisingly all of my 5 tubes give the correct band.

temps were 57, 59, 61, 63 and 65C.

the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer

#2 T C

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Posted 12 July 2009 - 01:44 AM

Its Okay

The smear could be coz of high template, just cut yr band of interest and proceed.

Best,
TC

I just received new primers so i ran a gradient PCR to determine best annealing temp.

surprisingly all of my 5 tubes give the correct band.

temps were 57, 59, 61, 63 and 65C.

the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer



#3 jiajia1987

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Posted 12 July 2009 - 06:16 PM

The one that gives the brightest band is the one with the good annealing temperature. your purpose is to find the optimal annealing temperature right?

#4 hanming86

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Posted 13 July 2009 - 05:21 AM

Try reducing the amount of DNA you're using

do 1:10 and 1:100.

see if it helps
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#5 Adrian K

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Excellent

Posted 06 August 2009 - 11:03 PM

I just received new primers so i ran a gradient PCR to determine best annealing temp.

surprisingly all of my 5 tubes give the correct band.

temps were 57, 59, 61, 63 and 65C.

the only problem is that I have a lot of smear above the bands,....but nothing below it or no primer dimer


This happen to me before. what I do was reduce the annealing time and extension time, and the smear gone, at least it works for me.

Hope you are not running a "rush" electrophoresis (160w ??)...this might make the smear on top happen too...for my experience.
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