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PCR and Primers


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4 replies to this topic

#1 ani

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Posted 09 June 2003 - 02:06 PM

Hello,
can I have some advise please? I am having trouble producing a positive DNA PCR result, after working through everything that may be going wrong I think it may be because the primers are not viable. Is there a way to check if primers are working without using DNA in the PCR reagent mixture? Thanks Ani

#2 hula

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Posted 09 June 2003 - 09:30 PM

Hi Ani,

Many things could have gone wrong such as primers, DNA template, dNTPs, polymerase, cycling conditioins. You have to check everything by running control PCR reactions. I would suggest that you first run a PCR using known good primers. If you can get bands from your template DNA, that means at least your PCR system is working. How big is your expected size? If it's big, you have to use some long distance PCR protocols.

Hula

#3 yossi.b

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Posted 19 June 2003 - 11:58 PM

hi in unix there is a gcg program with the command of foldrna "file name"
the program check if the primer can fold on it self. check this before ordering a new primers the delta g need to be -1 and more. below -1 the primer has good chance to fold on it self
good luck yossi

#4 jonnyr9

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Posted 03 July 2003 - 06:27 AM

Hi there - I'd familiarise yourself with a few programs before sticking to just one. There is a list of them at MolBiol.Net (the bioinformatics links database) - see http://www.bioinform...520Design.shtml

I think Oligo 3 is probably the fastest -

Keep trying and you will succeed!

#5 fei

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Posted 10 July 2003 - 11:44 PM

maybe it's not the fault of primers,you'd better check out the system first




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