Cloning does not work
Posted 16 May 2011 - 12:05 PM
Posted 17 May 2011 - 01:30 AM
I am trying to create a complement strain of my mutant. Several trials have failed so far. Can anyone help me in identifying steps where I might be going wrong? What precautions should I be taking and what could be the reasons for my failures? Please advise.
You'll have to give the forum members a little more information in order to help you out
Posting your protocol would be a good start.
Posted 20 May 2011 - 09:48 PM
Thankyou for your reply. I am trying to clone a 800 bp PCR product into 4.2 kb pacyc184 vector. Did restriction digestion simultaneously with BamH1 and Sal1 for both at 37 degree C overnight. Then purified the cut insert and vector using PCR purification kit. Then did ligation at 1:3 ratio (vector: insert) at 16 deg C overnight.Control without the insert was also used. Did ethanol purification for the ligation mixture. Took 2 ul of purified ligation mixture and carried out electroporation using 50 ul of TOP10 competent cells. Plated on 34 ug/ml chloramphenicol plates.
I didnt get any colonies on any of the plates. I dont know where I have gone. Please give your suggestions.
Posted 21 May 2011 - 06:47 AM
These are the most likely problems, but there are potentially many more. We can't help unless we know the complete details.
Posted 22 May 2011 - 08:39 PM
Appreciate once again your earlier reply