发信人: rinoa (blue), 信区: Biology
标 题: Re: help on ligation problem!
发信站: The unknown SPACE (Wed Jul 25 18:19:59 2001) WWW-POST

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? But it doesn't make sense to me. Anyway, if
this is the what happened to your pcr, it's for sure that
the digestion and ligation won't work. For my work, I did a
couple PCRs later and finally got the right one. Still don't
understand why. I still suspect it is the problem of
primers.
TA vector will sure help you find the problem, good luck.



【 在 royluo (roy) 的大作中提到: 】
: Thank you for your help!!! So far, four people including you
: have suggested this way to me. I will try to find an appropriate kit.
:
:
:
: 【 在 Morphin (莫非) 的大作中提到: 】
: : 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.
:
:


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