RT-PCR

January 3, 2005 at 4:42 pm #281

Participant

I am using SuperScript First-Strand Synthesis System for RT-PCR. I performed the reaction with control RNA and it worked. I tried 4 different primer sets for 4 different genes. 3 of the primer sets which amplify smaller than 500 bp fragments are working but 1 primer set which should amplify a 2 kb fragment is not working. Are there anyone who faced with problems with moderately long product amplifications?

March 12, 2012 at 3:34 pm #110108

cathbr

Participant

Hi,

a lot of things could happen between the RNA and your PCR… If you give me more details I might be able to help
For ex:
the conditions that you are using for the PCR
if you are using specific or random primers for the RT
if you try to amplify the same products, or spliced varients,… with 4 different primers…
Are you sure that your 2kb fragments is within the cDNA that you created?
Did you try to increase the elongation time?
Did you try different annealing temperatures?

January 23, 2013 at 8:24 am #113422

bravebeaker

Participant

as far as i know and correct me if i am wrong, RT-PCR amplification products should be short, not too short but not too long, if I understood correctly you are amplifying a 2Kb amplicon I think that is very very long.

January 23, 2013 at 3:27 pm #113424

JackBean

Participant

why should it be short?

January 28, 2013 at 10:18 am #113443

bravebeaker

Participant

I am saying based on the kit I am using which is SYBR green by TOYOBO and also many qPCR protocols over the internet as well as papers indicate the use of a short amplicon between 80 to 200 bp… what do you think? maybe banuy’s experiment is different?

January 28, 2013 at 1:49 pm #113447

JackBean

Participant

First strand synthesis and qPCR are IMHO something different. Yes, for qPCR one should have only short amplicons, but I don’t see reason why should you amplify only short pieces during reverse transcription.

January 29, 2013 at 1:19 am #113449

bravebeaker

Participant

Now I see your point, but in my case I use the primer mix that comes with the RT kit by Takara..

January 29, 2013 at 11:39 am #113452

JackBean

Participant

random hexamers?

January 30, 2013 at 4:27 am #113459

bravebeaker

Participant

yes

February 1, 2013 at 11:18 am #113468

JackBean

Participant

in such case you will have mixture of random lengths and only the length of amplification step will influence the maximal length of your amplicons

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