leman at leman.org
Tue Oct 19 19:05:36 EST 1999
On 19 Oct 1999 13:53:46 -0700, william at neuro.usc.edu (William Sun)
>Does anyone have experience assembling a cDNA from a set of overlapping
>oligonucleotides? How long can I make the primer and how big an overlap?
>Can I anneal all the primers and vector together in one step? Any advice
>or reference would be greatly appreciated.
Yes, I've done it. Once I had difficulties amplifying a 2100-bp cDNA
(BTW, it was rat serotonin transporter -- Go Neuro! ;-)) I don't
remember what exactly was the problem but it worked when I assembled
the whole stretch from 3 PCR products. I am not sure what the overlap
was, but I think it was about 100-150 bp. In other words, my primers
A-1-F, A-800-R, B-700-F, B-1500-R, C-1400-F, C-2100-R (or something
like that). Then you start assembling the fragments together, for
example: mix fragments A and B and use primers A-1-F and B-1500-R; or
fragments B and C and primers B-700-F and C-2100-R.
I "attached" one fragment at a time and used only two primers per
reaction. I don't quite see how one could use "all the primers" in the
same reaction. The rule of thumb is to use as long fragments as
possible to avoid "over-PCR-ing".
Now, since I am no longer completely ignorant in the mol-bio field, I
would dare to propose (entirely theoretically!) an alternative method:
use Klenow to assemble overlaping fragments and subcloning for
Well, these are just my 2 ¢. It's been almost 5 years since I did this
assembly, so don't sue me if it doesn't work ;-)
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