发信人: golem (毛毛雄东方必败), 信区: Biology
标 题: Re: help on ligation problem!
发信站: The unknown SPACE (Mon Jul 23 20:13:44 2001) WWW-POST

my experiences:
this year I tried cloning 4 genes, now only succeeded in 3.
The bad thing is I chose XhoI as one enzyme site for all
those genes, which turned out to be a big mistake. For some
reason, according to an experienced researcher in our lab,
XhoI itself isn't a good enzyme, star activity would be
found often.
For the 3 genes I made progress on, I screened about 200
clones for each construct, the positive rate is
extraordinarily low. 2-3/200 colonies.
Now I am still working on the 4th gene. If I failed this
time, I will change my primer to introduce another enzyme
site than XhoI.

So my solution for your case is:
1. use far more than enough PCR product to do the ligation.
e.g. a tube of product for a ligation.

2. screen at a large amount, e.g. 50 once.

3. use dephosphorase to treat vector to lower vector gay
activity.:)

4. change your primer for another enzyme site.

good luck.



~{!>~} ~{TZ~} Morphin (~{D*7G~}) ~{5D4sWwVPLa5=~}: ~{!?~}
: I am pretty sure that the enzyme digestion is the key point (3-nt-overhang i
: s way too short!) becasue I had the same problem a couple of months ago.
: Just subclone it into TA-vector (Invitrogen) and do what you should do.
:
:
:

--
一个人只拥有今生今世是不够的,他还应该拥有诗意的世界.

※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: hjpatt-136.umd.]