发信人: coconut (可可), 信区: Biology
标 题: Re: 谁有sequence cosmid的经验？
发信站: The unknown SPACE (Wed Jun 28 22:18:42 2000), 站内信件


I don't have the protocol w/ me, but I believe that you can look
it up in the journal Biotechniques. It is a quite reliable method
which is basically:

1. get a set of degenerate primers, usually designed specifically
for your organism.
2. design a specific primer which binds to one end of your DNA.
3. do 3 rounds of PCR, 3 minute elongation time, 10 minute extention time.
i) very low anealing temperature (35C?) for 25 cycles. take 1 ul,
do a 1:20 (or 1:100?) dilution and use 1 ul as template for the
next round
ii) slightly higher anealing temperature (45C?) for 25 cycles. take
1 ul of 1:20 dilution
iii) fairly high anealing temperature (50C?) for 35 cycles.
4. run all three PCR samples on gel. do gel purification to bright bands
on the third round of PCR, so you probably want to run a 100 ul reaction
on the last one.
5. sequence w/ the specific primer.

you get the idea.

【 在 roche (晚安！) 的大作中提到: 】
: don't know what is that.
: Can u tell me in details? Thanks a lot
: 【 在 coconut (可可) 的大作中提到: 】
: : Tail PCR is a good method.


--

百事皆可可，一心独悠悠

※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 169.237.218.28]