RT-PCR problems

Mr Steven Ennion steven at rfhsm.ac.uk
Wed Nov 1 12:16:57 EST 1995


>I'm having a problem with my RT-PCR reaction.  I have done the RT three 
>times and still no PCR products.

>I start with about 2ug total RNA (in 16ul depc H2O) and I know this is
>good quality stuff.Heat the RNA to 80 C for 3 min, place on ice for 3 min.
>I add (in order, on ice) 1.5 ul dNTP (10 mM each), 1 ul of 100 ng/ul oligo
>dT adaptor [primer 34 mer, 17-18 of which are dT), 6 ul 5X buffer (gibco),
>3 ul 0.1M DTT (gibco), 2.5 ul of superscript MMLV (Gibco).  Incubate at 37
>C for 1 hour, and use as template in PCR. 

>I run the PCR reaction along with a control template of a clone that 
>gives the right product, so I know the primers work fine.

>I have also done this RT-PCR before, and it worked fine.

>Is it wise to treat the cDNA with RNase H  or RNase A?  Is there 
>anything else I should Do?  

>Please help! 

>Mark Fry                                 

Dear Mark,
I use the following with good reproducible results,

10ug total RNA in 14ul DEPC water... heat 65 deg C for 5mins .. cool on ice
take 12.5 ul of this and add to  the following mix:
0.5ul RNase guard
5ul 5x RT buffer (500mM Tris Hcl, 600mM KCl, 100mM MgCl2 pH 8.15 at 42 deg C)
0.5 ul of oligo dT adaptor primer (500ng)
0.5 ul 1M DTT
5ul dNTP stock (5mM each in stock)
1ul RAV RT enzyme (Amersham) (15 units)

incubate 42 deg C 2 hours..
use 1ul of this first strand cDNA reaction for PCR .

Possible problems with your protocol could be..
1) Evaporation of yor volume of RNA during heating... thus 16ul could end up 
as 12ul in the final reaction and disrupt the buffer concentration
2) Your RT buffer may already have DTT in it so you may not have to add more


hope this helps



steve






     



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