I need help on Ligations
akravetz at MO.NET
Tue Apr 11 12:41:24 EST 1995
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
My ligation is using the Rapid Kit also by BM because we don't have a
water bath that can hold 16C in the cold room. The conc. of the two are
Insert 90ng/ul and Vector 500ng/ul.
I am trying to achieve a 3:1 molar ratio but to do that, requires more
than the amount of buffer required, so I have to EtOH precp 200ng, Not
I did a control of vector alone and also got no colonies, so that means
the vector is either cut and not transforming or not there and not
transforming.. BTW, these are LB-Amp plates.
I am stuck and don't know what to do. I am going to retry it but instead
of a molar ratio, I am going to use a mass ratio. if anyone has any
hints, let me know. I am at a loss.
St. Louis, Missouri
akravetz at mo.net
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