发信人: netbuggie (中原一点虫), 信区: Biology
标 题: Re: help on ligation problem!
发信站: The unknown SPACE (Sun Jul 22 22:47:15 2001), 站内信件

use NEBuffer2+BSA, digest enough amount of PCR product O/N. i do not know
if you add some nucleotides for buffer purpose. if it is for mutagenesis,
or expression of certain gene, maybe difficult. however, check with BioLab
for Hind3, even 12bp sequence (6bp as buffer flanking) could not be completely
digested O/N. for Xho, it is better, about 75%. so the 1st thing you should
do is to check your design, order new primers if necessary. they are so
cheap nowadays. actually, since it is only 150bp. i suggest you use pT7
Blue3 blunt end cloning kit to clone it into the blunt vector and sequence
it. if you do not wish to have mutation later, better start here, pay
attn to the PCR conditions. and by doing so, you can double check the primers.
for that vector, it is the same since they are so close. you may setup
a single digest (i prefer Hind3) to check star activity, i am afraid too long
digest will destroy the stick end, and avoid buffer contamination as well.
btw, your procedures were too complex, and you are running risk of lost the
DNA or destroy it. if i were you, PCR first, run gel (run all of the product),
gel purification (Qiagen kit), use a little bit for quantification. setup
30ul digest of H3/Xho (10U/ug, normally 15U/ug better), O/N (single digest
for ctrl). phenol/chloroform extraction, ethanol precipitation (since it is
150bp, -80 1hr). spin hard, air dry. for vector, you can dissolve the pellet
in larger volume. for insert, for 1ul T4 ligase, normally use about 100ng-
1ug, it works. so you should have rough idea of the concentration, set molar
ratio of insert to vector at about 20. take enough insert and set up 10ul
ligation, 16C O/N. use 2ul
for transformation of good-quality competent cell. hmm, what else, use fresh
Amp/LB plate.


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