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conversion of total RNA to cDNA


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#1 shankares

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Posted 09 February 2011 - 12:03 AM

Hello everyone, I need help with my PCR. I am new to the PCR and my aim is to detect the expression of specific genes by RT-PCR. I have enough total RNA (detected by gel electrophoresis, nanodrop) but after trancription I cant detect any cDNA. I dont know where I go wrong since I add all the reagents (random primers-70 C for 10 mins followed by 5 X buffer, 0.1M DTT, dNTPs and superscript II- 42 C for 50 mins).I would be happy if someone could help or give suggestions.

#2 BioMiha

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Posted 09 February 2011 - 01:10 AM

Do the PCR with the cDNA and I bet you will get bands. You don't explain how exactly you detected the cDNA, but since the cDNA is single stranded and therefore behaves much differently than dsDNA, you might just not see it.

#3 shankares

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Posted 09 February 2011 - 01:23 AM

I did RT-PCR and detected no amplification whatsoever. I also ran a gel and detected no visible bands. So I assume no cDNA

#4 BioMiha

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Posted 09 February 2011 - 06:28 AM

What kind of primers did you use for the PCR? You said you used random primers (=hexamers?) for the RT step, what kind did you use for the PCR step? You could try with oligo dT instead of the random hexamers.

#5 tea-test

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Posted 10 February 2011 - 04:42 AM

how did you dilute your cDNA after RT? undiluted cDNA reaction mix is a potent PCR inhibitor.
tea-test: The artist formerly known as Ned Land

#6 shankares

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Posted 10 February 2011 - 04:52 AM

The cDNA was diluted with DEPC water.

#7 ElHo

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Posted 11 February 2011 - 03:08 AM

Try amplifying a housekeeping gene (b-actin, gapdh) using a set of primers already established in your lab. Use cDNA from another lab member that has already worked before as a positive control.

#8 vidz

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Posted 24 March 2011 - 01:58 AM

Use oligo dT primer. Heat at 70 degrees for 5 min and then add your RT reaction mix and DTT solution. I did it like that and got bands on agarose.

Edited by vidz, 24 March 2011 - 02:00 AM.





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