phenol/chloroform of purified PCR product after RE digestion - what are we trying to purify? (Jan/26/2006 )
i have already posted this question in the pinned thread but just wanted some ideas more quickly since im digesting now.... 
if i have purified PCR product from the gel with low but enough recovery and I am digesting with enzymes....so can i just heat diactivate them and go on for ligation? why should I phenol/chlorofom if there are no proteins, lipids, RNA etc....? I will only lose more DNA...
what do you think?
Hi Kathy,
The biggest problem is the salt concentration of the reaction, if as you say you have got low recovery then you would be using considerable amount of digest in the ligation. The reaction conditions of the digest will then add to that of your ligation salt, pH etc. and probably disrupt the ligation.
Shouldn't really be a problem if you are only using a small amount of your digest in the ligation, though.
Good luck,
Scott
The biggest problem is the salt concentration of the reaction, if as you say you have got low recovery then you would be using considerable amount of digest in the ligation. The reaction conditions of the digest will then add to that of your ligation salt, pH etc. and probably disrupt the ligation.
Shouldn't really be a problem if you are only using a small amount of your digest in the ligation, though.
Good luck,
Scott
thank you Scott for your reply, i have had the problem because of high conc of ligation buffer in electroporation...therefore im concentrating my DNA with ethanol ppt.....ill ligate around 4ul of DNA....and 2ul of vector so in 6ul of ligation buffer.....does it seem ok?
It really depends on the concentrations of your insert and vector. It's best (though not always necessary for success) to calculate the molar ratios of your vector to insert ends and base your ligations off of that.
Hope that helps,
Hank
the ratio will be around 1:3 vector to insert....cant have higher ratio because dont have much insert... just i have electroporated but ms was 3.3ms....i think too low... anyway have to wait for colonies tomorrow if any....