cDNA synthesis.

[Anton Scott Goustin]

peter.nilsson at klinkemi.umu.se (Peter Nilsson) wrote:
>Hello,
>I am currently setting up cDNA synthesis to do a cDNA bank from mouse
>spinal cord. To be able to estimate the nummber of mice needed I need
>to know what yield of cDNA one could expect from each microgram of
>mRNA. Anybody know that? Also greatful for reccomendations of kits for >cDNA synthesis.
>
Most reverse transcriptases will convert 40-50% of the mass of poly(A+) RNA into cDNA, if you are using oligo(dT) as template.  Thus, 1 µg of
high quality poly(A+) RNA will yield 400-500 ng of cDNA.  This is alot of cDNA.  The last cDNA library I made, I got ~500,000 recombinants (in lambda gt11) from the packaging of 40 ng of ds cDNA.  I did not use a kit.  Either SuperScript MMTV RT or AMV RT will work.  Convert the RNA:cDNA duplex into dsDNA using the Gubler-Hoffman reactions (RNAse H,
Klenow).  Assure that the dsDNA is blunt using T4 DNA polymerase in the
presence of 250 µM dNTPs.  Add linker-adaptors, such as the NotI-EcoRI
set from Pharmacia, ligating a >100-fold molar excess.  If you want very long inserts, size the product on Sepharose CL-4B, keeping only the >1.5 kb fractions.  GeneClean the final product and ligate into lambda arms; package with an extract like those from Stratagene.  You can get hellish numbers of recombinants easily, 1 million or more if you need them, from 100 ng of ds cDNA.  With many of the lambda vectors, you can purchase
PCR primers (in the L and R lambda arms) which allow you to <<pop out>>
the cDNA inserts easily for subcloning, sequencing, etc.  If you want to use a kit, you can't lose by Pharmacia!

Even the worst tissues will yield >200 ng of total RNA per mg of tissue.  Unfortunately, that is only ~6 ng of poly(A+) mRNA.  You absolutely must
have good pure poly(A+) RNA if you want to succeed.   That is the bad
news.  There are quicky kits to select poly(A+) RNA direct from tissue
lysates, using magnetic beads (Dynal for example).  The big problem is
how much poly(A+) RNA the tissue holds.  Some cell types, like resting
lymphocytes have short A tracts in their RNA and very little mRNA per g
of packed cells.   That's why a total RNA method like RNAzol can be useful if tissue amount is limiting....