6.3Kb mRNA reverse transcription

Sébastien Vigneau via methods%40net.bio.net (by sebastien.vigneau from gmail.com)
Wed Jul 14 09:16:49 EST 2010


Hi,

I have never tried to reverse transcribe on such a long distance but, from
my experience on regular RT, SuperscriptIII works better than SuperscriptII.
In addition, based on extensive testing ~3 years ago by a colleague from my
former lab, "LA Taq" from Takara was the best polymerase for the
amplification of long (5-10kb) fragments.

Sébastien



On Tue, Jul 13, 2010 at 5:32 PM, Peter Ellis <pjie2 from cam.ac.uk> wrote:

> On 13/07/2010 16:16, B.Rama chandran wrote:
>
>> Dear all,
>>          I'm trying to make  6.3kb reverse transcript from mRNA. I tried
>> both poly dT primer as well as gene specific primers for reverse
>> transcription. But I didn't get 6.3kb product. So tried to amplify 'C'
>> terminal half (3.1kb). But I didn't get that also. I have used Superscript
>> II fro reverse transcription and Phusion polymerase for PCR. If anybody
>> have
>> any suggestion please give me.
>>
>
> Without more information, we can't give you specific help.  Here are some
> possibilities.
>
> 1)  Your RNA may be degraded - try a formaldehyde agarose gel to check the
> integrity of the sample.
>
> 2)  There may be a problem with the PCR reaction: run a control PCR on a
> working template (e.g. plasmid-specific primers on plasmid DNA) to check
> that your PCR enzyme / buffers / mastermix is working.
>
> 3)  There may be a problem with the cDNA synthesis: run a control RT-PCR
> using primers for a ubiquitous gene such as beta actin.
>
> 4)  Is your gene of interest actually expressed in your mRNA sample? Can
> you amplify short fragments, say ~200 bp in length?
>
> Peter
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