I need help on Ligations

Pablo Tebas tebas at visar.wustl.edu
Thu Apr 13 22:13:52 EST 1995


In article <3mef05$4ct at Twain.MO.NET>, akravetz at MO.NET (Andy Kravetz) says:
>
>Okay, I pouring out my heart and soul because I can't get the !@#$$ thing 
>to work. So I will ask folks out on the net. I am trying to clone a 400bp 
>insert into a 5Kb pMON vector. It is not working and I can't figure out 
>why. Here are some facts and maybe y'all can help me. The insert was PCR 
>amplified to have the two restriction sites that I need, Nco1 and EcoR1. 
>Then I gel-purified it using the phenol chloroform method. The vector and 
>the insert were cut prior to gel purification and both were done the same 
>way. Using B-M enzymes which say the two can use the same buffer at 100 
>percent efficiency. 
>


Many people have give you good advice. I would tell you:

1. Several people that I know have had problems cloning PCR products with Nco 
at one of the ends. I do not know the reason, but probably as somebody has sugested,
is related to the number of bp needed to the end, or other reasons...

2. If you are using Taq, which puts overhanging A´s at the ends, you can clone
the fragment using a vector with overhanging T´s (promega pGEM-T, or you can make it).
This vector allows blue-white selection and is easy to use. When you have 
this intermediate plasmid, then digest with Nco and Eco and use as much as you
want. Nco should not have any problem cuting a plasmid

3. If you dont have pGEMT (and do not want to buy it), just linearize any vector
with a blunt end enzyme and incubate with Taq and dTTP for 1 hour at 70 degrees. 
Precipitate it and use it instead of pGEMT.


Good luck


Pablo Tebas
Washington University
St. Louis



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