RT-PCR

[Martin Kennedy]

In article <9210090425.AA07044 at cscgpo>, Klaus.Matthaei at anu.edu.au writes:
> G'Day Netters
> 
> I have some questions regarding PCR from RNA.  The standard method is to
> use Reverse Transcriptase and oligi-DT to synthesise cDNA from total RNA
> followed by standard PCR.  Another method is to use the dowmnstream primer
> for the cDNA synthesis followed by PCR.  More recently Amersham has brought
> out a taq polymerase called Tet-Z which has both RT and polymerase activity
> (indeed Amplitaq from cetus does this also).  The reverse transcription and
> the PCR is therefore done in the same tube in the same set of cycling
> reactions, the first one being a 15-30 minute RT step followed by standard
> PCR.  We have also tried these methods (when I say we, read 1 honours

 (STUFF DELETED)
> 
> Any help will be appreciated.
> 
> Cheers, Klaus
> --------------------------------------------------------------------------
> Klaus Matthaei



Klaus:

If you are just looking for RT-PCR methods that work rather than direct
experiences with Tet-Z, I do quite a lot of RT-PCR for isolating probes
and for anchored-PCR and I use the following:


First strand cDNA synthesis:
		
		5ug total RNA (or 1-2ug polyA if available)
		1ul RNasin 
		H2O up to 13ul final vol

Incubate 65oC for 5 min, then -> ice		

Add:		1ul of oligo dT (or GSP) @ 0.5ug/ul
		1ul RNasin
		4ul 5 X buffer + dNTPs
		1ul Reverse transcriptase

I use total RNA (poly-A not necessary) isolated by phenol/SDS extraction; 
RT primed with either oligo-dT or a gene specific primer, with LifeTech-BRL 
Superscript and the buffer supplied with this enzyme(but you must add in all
four dNTPs).  

Incubate 42oC for at least 1 hour.

Add water up to 100ul final vol, and heat at 70oC for 10 minutes.

This is ready to use in PCR - just take 5ul into a 50ul PCR rx, and go for it. 
No need to remove the diluted primer.
		
If you want to do anchored PCR or clean up the cDNA further, then I do 3 washes
through a Centricon-30 spin dialysis cartridge, with 1ml H2O each time.

Hope this is useful,

Cheers,
Martin