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Some primers dose not work


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5 replies to this topic

#1 Mohamed samir

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Posted 31 March 2013 - 02:29 PM

Iam pretty confused to ask this question, Has anybody an experience with non working primers after being received from Qiagen. The point that I received primers but it dose not works out. what may be the causes ?

#2 Tabaluga

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Posted 31 March 2013 - 02:53 PM

Did you try several annealing temperatures (i.e. gradient PCR) ?

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 almost a doctor

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Posted 01 April 2013 - 01:57 AM

Can you tell us what you've done, and why you've concluded the primers don't work?

Also, are this newly designed primers (ie. never tested) or have they been shown to work before?

The more you can tell us (detailed protocols you've used), the better we can help you.

#4 Mohamed samir

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Posted 01 April 2013 - 08:54 AM

Actually my colleague did real time PCR and he find nothing. Can he do several cycle with various annealing temperature, is this the gradient PCR u mean ?
The protocol, he looks for the expression level of iRG1 gene in the A549 cell line. He ordered the primers without designing them . the primers came directly from the company.

#5 Tabaluga

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Posted 03 April 2013 - 09:18 AM

Find your optimal annealing temperature with a gradient on a normal PCR, not real time. The PCR machine can build up a temperature gradient, so that in the same run each row of tubes is run with another annealing temperature. Also see here: http://www.protocol-...posts/4497.html
Then check on the gel: Is the band in the correct size ? If yes, at which temperature did it work best ?

I think almost a doctor meant the detailed PCR protocol (how much of which reagent etc.)

What do you mean by "he ordered the primers without designing them" ? Of course they came from the company, but he must either have looked the sequence up in some publication where they were used or designed them via PrimerBLAST or Primer3 etc. for the company to synthesize them...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#6 mdfenko

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Posted 04 April 2013 - 04:39 AM

What do you mean by "he ordered the primers without designing them" ? Of course they came from the company, but he must either have looked the sequence up in some publication where they were used or designed them via PrimerBLAST or Primer3 etc. for the company to synthesize them...

i take it to mean that standard primers were purchased.
talent does what it can
genius does what it must
i do what i get paid to do




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