Help with RT-PCR!!! What's wrong with my primers?

Mark nospam at nspam.com
Fri Jul 23 07:51:40 EST 2004


Miao wrote:
> Hi,
> 
> I use invitrogen one-step RT-PCR kit. None of my 5 pairs of primers
> give me any visible band! I did a lot of control, so I am sure that
> the kit still works, my RNA template is good also.
> 
> I even tried those primers to amplity from plasmid (containing my
> gene's cDNA), but none of them worked!
> 
> All of them look OK, annealing temperature not very different,
> reasonable GC content, length 20-25 nucleotides.
> 
> Could somebody help me? I am so dissapointed!
> 
> Miao

You might need to be more specific with the type of controls you 
have tried. It would seem that your problem (one of them at 
least) is not with RT, but with the PCR itself (since the plasmid 
template gives no product in PCR). The first place to start would 
be to manipulate the 'usual' PCR parameters such as annealing 
temp, Mg concentration, the use of additives such as DMSO etc. 
Without more details from you as to what you have already tried, 
it is hard to be more helpful




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