Post-RT-Rxn.. - (Dec/18/2006 )

Hey guys,

I know my doubt is stupid..

Can anyone visualize cDNA after RT rxn, on agarose gel.. If CANT, why NOT!! b/c cDNA is double stranded, aint it!!!

Help is needed!!!!!

-Vick-

How to fish-out a low copy number gene from the total RNA by using RT-PCR..

Can anyone help!!!

-Vick-

use a specific primer.

-mdfenko-

When you do RT, all(or most) of the mRNA present will be reverse-transcribed to cDNA. So the length can very starting from base pairs to kilo bases, thats why you would see a smear if you run cDNA on agarose gel..
(Check the attached picture , got by googling..)

-Calvin*-

So is there a way to confirm that RT was successful and there was cDNA??

-tictactoe-

QUOTE (Calvin* @ Dec 20 2006, 12:24 AM)

Thank you so much for answer. When asked my lab mates almost everyone said my doubt was stupid and said we can never see it. Itried googl but couldnt find anything,,

Well, now I've got the picture prove it. Thanks once again..

-Vick-

QUOTE (mdfenko @ Dec 19 2006, 11:34 PM)

use a specific primer.

I've been trying that for the past 6 weeks, no luck!!

10 different annealing temperatures,
4 sets of primers.....
3 different GC melt mixes,
2 different RT-PCR buffers,
2 different Taq's

All in vain..

If you can tell me what I'm doing wrong, I'll be really glad..

-Vick-

QUOTE (Vick @ Dec 20 2006, 03:47 AM)

have you confirmed the existence of your mrna by northern analysis? q-pcr?

what do you use for reverse transcription (oligo-dT, random hexamers, nonamers, dodecamers)?

-mdfenko-

QUOTE (mdfenko @ Dec 21 2006, 01:41 AM)

QUOTE (Vick @ Dec 20 2006, 03:47 AM)

have you confirmed the existence of your mrna by northern analysis? q-pcr?

what do you use for reverse transcription (oligo-dT, random hexamers, nonamers, dodecamers)?

Well, I'ven't confirmed the existence of my mrna by northern analysis or q-pcr!!, that looks like a good idea.

I've used Oligo-dT once and random hexamers once, but no luck again..

-Vick-

do you have any knowledge of the sequence from the poly-A? if so you may be able to selectively reverse transcribe your m-rna by using oligo-dT with the complement of your sequence from the poly-A. (i don't know if this would work, just a thought)

-mdfenko-