PCR Direct Sequencing

eric c. anderson anderson at pharmdec.wustl.edu
Sat Jul 16 09:59:56 EST 1994


In article <1188 at biosys.apldbio.COM>, inherit at apldbio.com (Inherit Account)
wrote:
> I have a question about PCR Direct Sequencing. Do you think what is most important thing to do that? 
> In PCR stage, the product is amplified well with no extra band, but sometimes I couldn't get good sequence result. Noisy. What is wrong?
> 
> And I try TA cloning but it need more time. I hope, it is possible, do PCR Direct Sequencing for every templates.
> 

i've been working (passively) on a protocol to do that for months.  despite
changing almost every possible parameter of the situation (template and
primer concentration, cycling times, clean up, etc.) nothing seems to work.
 from your mail address at ABI, i'm surprised you asked this question. 
it's been my impression that the party line at ABI/PE is that this doesn't
work.  i don't really know why it doesn't work, but i'm beginning to agree
with them.

so far, TA cloning is the fastest and best way that i've found to get the
sequencing to work.  it takes way more time than i would like, but i think
it's the best choice.

sorry,
eric
-- 

BIG AIR...

NOT BIG PANTS!!!
---------------------------------
eric c. anderson
anderson at pharmdec.wustl.edu
660 s. euclid box 8103
washington univ. school of medicine
dept of molecular biol. and pharmacology
st. loser, misery 63110



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