I need help on Ligations

[Andy Kravetz]

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. 

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 
Fun. 

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. 

thanks all

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Andy Kravetz
St. Louis, Missouri
akravetz at mo.net