small fragment library construction

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Sep 23 11:04:35 EST 1999



"Mr. M.J. Lush" wrote:

> In article <37EA413D.66E69651 at uni-konstanz.de>,
> Frank O. Fackelmayer <Frank.Fackelmayer at uni-konstanz.de> wrote:
> >Hi all,
> >I´m collecting ideas on how to make a library of short DNA fragments in
> >an expression vector. I have a 2.7kb cDNA fragment that I want to split
> >into many overlapping fragments in the 30-80 base pair size range.
>
>         Gak!  30-80bp is very small....  If you up for a slightly
> mad idea you could run a  'short oligo PCR',  running 2 to 4
> rounds of PCR using random n-mers (I wouldn't like to say which would be
> the best size 6-7bp?) with lots of target cDNA.
>
>         In the first round of cycling the n-mers would anneal at random
> to the cDNA and extend,  in the second round of PCR they would anneal to
> the cDNA and the 1st round products all of which which would be truncated
> producing much shorter dsDNA products,  additional rounds of cycling would
> yield even shorter products.
>
>         Run the product out on a strong agarose gel (Metaphor?) and
> cut out and purify the 30-80 bp section.
>
>         The main problem I suspect would be keeping the oligos annealed
> at Taq's working temperature,  I suspect you would be better off running
> the reaction the old fashioned way with DNA Pol and replacing the heat killed
> enzyme at each cycle.
>
>         If anyone trys this please let me know if it works,  I suspect
> I may have use of the approach at some stage!
> --
>
> Michael

Hi Michael,

thanks for your quick answer. Yes, that idea came to me also, and I have already
tried it for two weeks now with several different primer constructs. However, it
didn´t work at all, because I never got significant amounts of product. Primers
were random 6mers with an additional restriction site on the 5´ side. Cycling
manually with Klenow also didn´t improve the result, even with extension
temperature of 10°C or 25°C.
I don´t know exactly what goes wrong, because in principle it should work well.

Best wishes,
Frank




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