Cloning does not work - (May/16/2011 )
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.
snehaSK on Mon May 16 20:05:27 2011 said:
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.
Clare
Hi Claire
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.
Thanks
Sneha
There are many potential problem areas. First, I would avoid using SalI as an enzyme for cloning, if possible. It is a known troublemaker. But more likely your difficulty is in other areas. Some specific ones include the design of your PCR primers -- do you include extra 5' bases beyond the restriction enzyme site? How many? You should test the competence of your cells by making serial dilutions of pACYC184 down to the 10 pg/ul level and electroporate them. You should get 10^9 to 10^10 colonies/microgram with electroporation. Very few people purify the ligation product following ligation. I'd recommend against it. Just add 1-2 ul of ligation product to your cells prior to electroporation. Do not use "quick" ligase, which is inhibitory for electroporation.
These are the most likely problems, but there are potentially many more. We can't help unless we know the complete details.
Thankyou very much for your reply. I added three bases prior to the restriction site in the PCR primers. I will try electroporating using just the vector. If you let me know what kind of further details you want I will be willing to provide.
Appreciate once again your earlier reply
Sneha