In my lab,we are trying to construct a control plasmid. So we have to remove the insert gene which has two restriction ends:one is XhoI and other one is Hind III.So after double digestion we want to ligate these ends and construct an empty vector.
But the problem is in these ends, there is no complementary bases to ligate.
Xhol site:
5'-C/TCGAG-3'
3'-GAGCT\C-5'
HindIII site:
5'-A/AGCTT-3'
3'-TTCGA\A-5'
So do PCR by designing primers having the restriction site of these two enzymes along with extra bases those having complementary bases to each other will it be a solution for this?
Hi Deepak,
If you want to self-ligate a plasmid digested by enzymes producing incompatible overhangs, you can make those ends blunt and continue your work. To remove the 3' overhangs or fill up the 5' overhangs you can use the Klenow fragment.
please follow the link below:
https://international.neb.com/products/m0210-dna-polymerase-i-large-klenow-fragment#Product%20Information
Hi Deepak,
If you want to self-ligate a plasmid digested by enzymes producing incompatible overhangs, you can make those ends blunt and continue your work. To remove the 3' overhangs or fill up the 5' overhangs you can use the Klenow fragment.
please follow the link below:
https://international.neb.com/products/m0210-dna-polymerase-i-large-klenow-fragment#Product%20Information
you could make 2 complementary oligos with the cut sites and a few extra bases to assist cutting,anneal them to make double stranded and cut both ends off then anneal the cut fragment into your plasmid if you do not mind having a very short sequence between the 2 cut sites
if you do what you have mentioned here, those 2 restriction enzyme sites will be altered.
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