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
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#1
Curtis
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Posted 11 July 2009 - 07:13 AM
#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
The smear could be coz of high template, just cut yr band of interest and proceed.
Best,
TC
Curtis, on Jul 11 2009, 08:43 PM, said:
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
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?
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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
do 1:10 and 1:100.
see if it helps
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#5
Adrian K
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Posted 06 August 2009 - 11:03 PM
Curtis, on Jul 11 2009, 11:13 PM, said:
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
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.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
