6 replies to this topic

[Curtis]

I synthesized cDNA from a viral RNA by random hexamers, and I can easily amplify 1.4 and 1.7 kb size genes, but the 6.7 kb gene won't amplify.

I thought maybe my reverse transcriptase doesn't synthesize cDNA that long? or could it be that my RNA degrades too fast that all fragments are smaller than 6.7?

Edited by pcrman, 13 February 2012 - 02:21 PM.

[Curtis]

Bob1? you there?

[bob1]

Having had a quick look at a superscript manual, it seems that you should be able to get cDNA up to 15 kb in length.  Perhaps you are having some issues with where the random hexamers are binding.  Could you try oligo dTs or specific primers (if the sequence is known)? You may have better luck with that.  You may also want to  try a longer incubation time to allow time for the longer product to synthesise fully.

You may also need to play with the ratio of hexamer to RNA to get an optimal yield for longer products, but I'm just guessing here.

[Curtis]

cool, thanks,

Oligo dT I can't use, because it is a viral RNA that doesn't have the polyA tail.

I use a Fermentas Kit, It is a new product that they claim it can synthesize up to 20 k. It is called RevertAid Premium cDNA synthesis kit.


so according to what you are saying I need to :

1- use gene specific primer. (Actually yesterday I did that but haven't run PCR yet. It is the first time I use GSP. I normally use random hexamers)

2- Incubate more. (ok, next time I will do that. I normally incubate 30 min @ 50C)

3- adjust the ratio between random Hexamer and template RNA. (ok)

[bob1]

That seems to be about it, I'm not sure how effective 2 and 3 will be, but they should help.

[phage434]

You can also make several pieces, using specific primers to each part of the construct, then put them together.

[Curtis]

put them together? but today I synthesized cDNA with a primer that binds to a region 100 b upstream of the gene. hope it worked. I will run PCR with it tomorrow and update this thread.

Edited by Curtis, 15 February 2012 - 07:49 AM.