RT-PCR and gels

Jim Hutchins hutchins at fiona.umsmed.edu
Wed Aug 2 10:55:27 EST 1995

: I believe it is possible to run RT then take 3 ul or so and run
: on a 1% agarose gel to verify you have a product. Correct?

I doubt whether this will work; you would need >1 ug of cDNA to
see a product, and if you are using specific primers this is 

: If not, then how can you check to see if your RT worked before 
: wasting reagents on PCR?

Yes.  The `standard' method is to `spike' the RT reaction, or a 
duplicate reaction consisting of 1/10 of the RT reaction, with
a small amount of 32P-labeled nucleotide (32Palpha-dCTP, e.g.).
Then, from the known molar amounts of each reactant, and making
assumptions about the size of your cDNA, you can calculate a
percent incorporation after separating the reaction from the
cDNA on a spun column (or better, two spun columns).  Most people
use Cerenkov counts to get a guesstimate of the percent incorporated.
In my hands, this number is very, very low.  For example, in a kinase
reaction, we expect 20-50% incorporation, but less that 1% is not
uncommon with an RT reaction.

Anyone else have any comments on this?  What do other labs get as
a percent incorp in a `typical' RT reaction?

Oh, I just remembered, some people use TCA-precipitable counts, which
is cheaper and easier than the spun column unless you already have
them around.

Hope this helps.

Jim Hutchins
Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
http://fiona.umsmed.edu/~hutchins/    ***    E-mail:   hutchins at umsmed.edu
``It became necessary to destroy the town in order to save it.''--American
infantry officer firing on Ben Tre, Vietnam, 2/8/68

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