PCR primers problem
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PCR primers problem
I study the expression of TNFa in porcine spleen cells. There is only two
papers who gives the sequence of primers to use. I tested these 2 primers
set. I got a signal for first three PCR reactions, but after (e.g. other
PCR reactions with the same cDNA) the PCR signal (band intensity) decreased
until it disappears.
I diluted the primers in sterile water. The primers were used at 0.25uM,
dNTP at 5mM, TAQ (Perkin Elmer) 0.75 units and with Perkin Elmer buffer.
Total volume : 25 uL. The PCR reaction, RTases and electrophoresis on
agarose are good since our positive control (human) are good.
Can we dilute primers in some buffer instead of water. If yes, which buffer
?
Is there a special task with porcine primers?
Does water have any effect on the primers?
Last update 22-Feb-2001, Rating Good of 4 votes.
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can i dilute stock solutions of dNTPs with TE buffer instead of water to make a working solutions of dNTPs.
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Iam doing PCR with long gen size (2.5) and my primer is too big around 40kbp, unfortunatly i have not got any result. So far i did change my TM from 55 to 70 but no band yet
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Yes - I agree. I always dilute my primers in TE (50mM Tris-HCl, 10mM EDTA, pH 8.1)
DD water, pH is slightly acidic, which may destroy primers in long term use.
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Yes - I agree. I always dilute my primers in TE (50mM Tris-HCl, 10mM EDTA, pH 8)
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Hi, by the way can anybody suggest a good supplier for primers? I am located in ontario, Canada
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Hi, by the way can anybody suggest a good supplier for primers? I am located in ontario, Canada
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Yes - I agree. I always dilute my primers in TE (50mM Tris-HCl, 10mM EDTA, pH 8)...if you have eg. 61.2nmol primer, resuspend the dry pellet in 61.2microlitres of TE, making a 1mM solution, which can be diluted to 10micromolar, or 10 pmol/microlitre solution by diluting 1:100 with water. If the PCR fails, or the primer stock is old, simply make up new stock. I have no problems with DNase in primer stock, but I get them from Genosys.
If you need help designing primers, there are 9 resources for primer design and analysis listed at http://www.molbiol.net and there's also a link to Virutal PCR in the new tools news section...
Jon
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You might try TE. We have had problems with degradation if the primers were in water and were stored in the fridge. The EDTA in TE slows degradation by chelating free Mg++ and so slows dnases.
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