TA Clonning

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Sun Sep 9 21:06:49 EST 2001


No true! Phosphorylated inserts, in most situations (yes, even in TA
cloning), is a plus.
The ligation problem is difficult to solve through such long-distance
comments and suggestions.
My suggestion, nonetheless, would be to repeat the whole thing with
properly cleaned PCR product. You (Peter; "Peter Guo"
<yuyuanguo at students.wisc.edu>) don't mention if and how the insert was
cleaned. The PCR product needs to be purified and isolated from the unused
primers and dNTPs for efficient ligation. Then there is the question of the
buffer used, the ligase, etc. 

However, the TA vectors from invitrogen have showed differential
efficiencies from time to time. The age of TA vector prep also seems to
have a bearing on the efficiency of ligation (although Invitrogen has said
that it is not true!). 

The ligation does not need to be at 4 C. For PCR products, which are to be
considered "blunt" for all practical purposes, are best ligated at room
temperature (22-24 C), but O/N at 16 C also should work. And, the ligation
is usually complete between 15min and 3h depending on the different
parameters involved. 

Finally, one should not lose sight of the fact that a ligation reaction is
only as good as the transformation step.  

Hiranya 

At 11:40 PM 9/9/01 GMT, Scott wrote:
>> I tried to clone a PCR product. However, after transform the Top10
>> compentence cells, I did not get clones. The PCR is good. I phosphoralted
>> the 5' end of the primer. I used Invitragen Company's kits. Ligation
>> condition is at 16 degree centigrade. I think it should be at 4 degree
>> centigrade. Is this correct?
>
>Check the manual for the TA cloning kit. I don't think it works with 
>phosphorylated inserts. At least that's what I seem to remember with the 
>TOPO-TA products.
>
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu

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