How to delete a domain in a gene? - cloned plasmid (Oct/06/2010 )
Curtis on Tue Oct 12 15:46:08 2010 said:
However if you check the link that I gave in my previous post their method is very quick also. They do 2 PCR steps. the second step self-ligates the PCR ends from first step. It sound very easier than ligation and phosphorylation!
Curtis~
The protocol you posted is not a ligating step but rather you are amplifying each side of your gene upstream and downstream of the desired deleted area and then using these two pieces as template in the second pcr to create the full length gene with the deletion. The two pieces do not ligate per se but they do have small overlapping regions so the polymerase can use both to create the final product. At the end of this step you are going to have a pcr product that you need to ligate into a vector as normal. Normally, when you digest a piece of DNA with restriction digests, it leaves a phosphate group on the end. You must have a phosphate on at least one end (insert or vector) in order to get ligation. Usually people use the phosphate on the insert (that is present after restriction digest) and that way you can dephosphorylate the vector to prevent recircularization. No phosphate, no ligation. But, it you order oligos and don't plan on digesting, you must add the phosphate with a kinase.
Hi Curtis,
its god speaking, directly from heaven ...no, i'm just joking 
The method that provided in your link is also known as stich PCR. The protocol sounds very easy but in reality it is not that straight forward as it looks like. I think re-circularization of a PCR product is faster and easier since you only need one pair of primers ...but thats my personal opinion ...other people may think different and might have different experience.
The thing about ligation and phosphorylation:
If you ligate for example a PCR product in a vector that was opend by a blunt-end cuter you do not need to phosphorylate since the vector will have the phosphate groups that are necessary to create the phosphodiesther bond. If you are just re-ligating a PCR product it is essential to phosphorylate, otherwise you will end up with NO colonies except the background-colonies created the template plasmid. Do phosphorylate even if it is not stated in the manual of Fermentas ...believe me and you will live happily ...in prosperity
Good luck!
Best regards,
p
Curtis on Tue Oct 12 15:46:08 2010 said:
However if you check the link that I gave in my previous post their method is very quick also. They do 2 PCR steps. the second step self-ligates the PCR ends from first step. It sound very easier than ligation and phosphorylation!
thanks so much guys, you got your tickets to heaven 