How to get GOOD rt-pcr bands - (Jul/09/2008 )

Hi...

Ive been isolating RNA from tooth pulp cells..and there are these few genes that i would like to amplify..but the RT-PCR reactions were not successful. i kept getting these unwanted bands in my agarose gel..

Here I've attached the gel pic.the band that i wanted is about 758 bp(alkaline phosphatase) while the band that i kept on getting is around 100+ bp.can anyone please help

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Are you sure your primers are specific and that your product really is 758bp?
What are your PCR conditions?

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I've been getting the primers from a journal..n ALP was amplified successfully from the tooth sample.n the expected size was 758 bp.I'm sure that the primers are spesific.my pcr conditions are;

1st strand DNA synthesis
42C, 60 min reverse transcription
99C, 5 min AMV/RT inactivation

2nd strand synthesis and PCR amplification
94C, 30 sec denaturation
54C, 1 min annealing
72C, 1 min extension

68C, 7 min final extension

4C (soak)

please help

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1. Did you try to blast the primers on the genomic/total cDNA sequence? May be worth checking specificity.
2. Is your extension step long enough? It may be worth trying 90-120 second extension.

Those were my ideas.

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Generally, I do not trust published primers.

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You are only kidding yourself... please stop posting such replies, people who don't know much might actually take you seriously.

now the reply to the topic... the same story happened to me a while ago, I found out new degenerate primers had been designed for one of the genes I investigate, I decided to try them out with the PCR setup from the paper, it wasn't working. I am a bit worried that some authors don't actually publish the PCR program their technicians were using hum... So you should use the different possibility out there to optimize your PCR young man. I did and now it's working fine.

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Hi..

thanks 4 d reply..guess u understands what I've been through..

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Hi..

so I guess d best way is to design d primers by ourself...right!

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