this is my first experience in overlap PCR, i must attach restriction enzim in my primer, and that'sprimer will use and ligase at plasmid. but the plasmid and my gen interest didnt have suit restriction enzim at the terminal. what should I do
select a restriction site that is in the plasmid and just add the enzyme site at the 5' end of the pcr primer. Add another 3 random bases outside this site again at the 5' end of this primer to ensure that the enzyme cuts. do not include the restriction site sequence when calculating the annealing temperature of this primer, The restriction site will be incorporated into all of the amplified product which can then be cut with the enzyme and the cut product purified. This can be done for primers at both ends of the amplimer
Since I was really impressed by ligase independent cloning, as opposed to the normal ligase dependent cloning I would highly recommend giving this a try. The idea is to open your vector with some restriction enzyme. Then you amplify your insert but you attach to your fwd and rev primers 15 to 20 bp of overlap sequence with your vector. Then you mix the purified vector and insert, add exonuclease III (NEB) and this generates big sticky ends. Now you just have to transform the reaction and the bacteria will repair all gaps. Works much better in my hands than normal cloning. Also works with multiple inserts. Just follow e.g. this protocol here: http://hanlab.xmu.edu.cn/Protocol/LIC%20protocol-梁耀极.pdf
Laurens Wachsmuth, your link is not opening. Please check and provide fresh link.
well, the Chinese characters seem to destroy the clickable link. Just copy and paste and it will work. But while we are at it: I just transform the reaction directly after step 9. Works great.
Hello Agus
I advise you designing a sequence of restriction enzime in your primers. You should realize that this sequence generate cohesives ends. In order to get better performance of ligation.
Regards
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