Re: help on ligation problem! - 未名空间MITBBS历史存档

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Re: help on ligation problem!

Re: help on ligation problem!# Biology - 生物学

n*e2001-07-22 07:07

1 楼

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.

l*k2001-07-22 07:07

2 楼

too long to read :)
I think it is possible pet20 have leaky expression, it may kill the cells.
in this case, I think novagen have a cell line that won't have leaky expression.
Instead of cloning disgested fragment, how about do blunt cloning using
novagen's perfect cloning kit? then cut it, at least you won't worry about the
short seq at the restriction site, after cloning,
with multiple clone sites on both side, you
have more choice to put it to pet20, though you have to worry about the problem

g*m2001-07-22 07:07

3 楼

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 g

r*a2001-07-22 07:07

4 楼

Then count me as the fifth. :)
Your problem reminds me one problem I had before. I kind
of did the similar things, PCR a short piece, about 140 bp.
After I subcloned it into TA-vector and
the sequencing, I found the gene was in the vector except
the restriction sites. Weird. Currently my labmate is having
the same problem, the restriction sites on the ends of
primers are not there although all other sequences seem
fine. Her piece is about 120 bp. Is it related with the PCR
of small pieces? B