vector dephosphorylation - (Sep/21/2013 )
I am using a antarctic phosphatase and a line in the protocol says, '' incubate for 15 minutes at 37 deg C for 5' extensions or blunt ends, 60 minutes for 3' extensions. ''
Does it mean, for 15 minutes it will remove 5' phophate groups and in a longer incubation of 60 minutes it will remove 3' groups (which i think is hydroxyl or is it phosphate too in some conditions?). i jjust want to prevent vector religation before i put the insert. Pls help understand this line in the protocol.
You all have always been very helpful, esp. veteran moderator, and am very thankful, thanks in advance again.
also after vector dephosphorylation i am planning to clean up my vector with a kit, should i just heat inactivate it as well at 70 deg c for 5 minutes as per protocol, am afraid if it will damage the vector, its pmirglo vector.. thanks.
No, the phosphatase is use to remove 5' phosphates. 3' phosphates are rare in biology. What they are discussing is the location of the 5' phosphate on a double stranded piece of DNA. When you cut with an enzyme such as EcoRI, it leaves a 4 base overhang, extended at the 5' end. This is dephosphorylated in 15 minutes. Similarly, a blunt cutter such as SmaI will leave no overhang, and will also dephosphorylate well. Unusual cutters such as PstI will leave a 3' overhang. These take more time to dephosphorylate. You can see why if you think about the relative accessibility of the 5' phosphate group in these cases.
Rather than dephosphorylating your cut vector, you might think about PCR amplifying the segment you need, then cutting to leave the correct overhangs. This removes most of the circular vector (especially if DpnI treated) and reduces background more than dephophorylating will.
thanks a lot , honestly this is very helpful. cheers..