RT PCR

[is5298mih]

I am interested in amplifying a 3.0 kb cDNA using primers whose Tm are 67 and 64 C. The template has GC of 55%. the cDNA smear on a gel shows a wide molecular range including the 3.0 kb molecular size fragments. The cDNA positive controls worked as expected.

My PCR was done using 0.5U Taq, 200 uM dNTPs, 200 nM each primer, 1x Buffer, and 2 ul of cDNA synthesized using a Super script II kit in a 50 ul Rx. My PCR programs were done separately in varying annealing temperatures: 94C 5′ x1, [94C 30", 40C/45C/50C/55C/62C for 30", 72C 10′]x30, 72C 10′ x1. I also tried touch down PCR from 50-65C annealing.

My results: at 55C, I was able to see a very faint band in the correct size.

Any suggestions?

e-mail directly:

ismaili@ccri.net

Thanks,

[LilKim]

hmmmmm.. somethine seems a little strange.

Are you running a control cDNA sample not prepared by yourself? Is there a chance that something has happened to you cDNA?

Do you have more than one PCR machine on… I know that this sounds krazy… but once I was having a PCR nightmare… and it was caused by the fact the block was not heating properly. It was like … everyhing worked one day and then NOOOOOTHING worked for a month. (thank God it was the machine and not me) I hate to make you paranoid; but, it happened to me… so save the aggravation and try somewhere else.

Also, are you DNTP’s ok? is your water, taq… etc. okay? Maybe you can try someone else’s reagents to see if that will work.

i wish you luck with … resolving this.
– kim

[shaikmohamed]

Hi,

If you get any expected size of band that is fine. Your PCR is working however, some thing happened to your reaction (cDNA, primer or dNTP degradations). Cut the band, extract the DNA and use 1 or 2 uL DNA for you nested PCR with new primers (old set of primers may work, you can check before going to order new one). Definitely, the nested PCR give more product of the gene. If works pls let me know.

Best Wishes
Shaik

[schmyly]

I learned something here!

[Author]

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