Dephosphorylation of insert - Dephosphorylation of insert (Jul/15/2010 )

Hi,
By mistake I added Shrimp Alkaline phosphatase to my insert digestion and not to my plasmid digestion.
My plasmid is ~7kb and insert is ~550bp, and they are digested with 2 different restriction enzymes HindIII and NdeI.
Should I go ahead and ligate and transform or is it not worth it?
Thanks

Cymbelline on Jul 15 2010, 12:54 PM said:

It depends, there are 3 options open to you

1. Add T4 PNK (polynucleotide kinase) and associated buffer to your insert. Incubate it for 30 mins or so at 37C (see instructions of PNK). PNK will add the lost phosphate. Once the job is done, remove PNK by heat inactivation OR column purification OR phenol-chloroform.

2. Put everything in and continue ligating. The vector still has phosphates although the insert is now dephosphorylated, so you can still get your plasmid. As you have cut the vector with two RE, the overhangs of the vector are incompatible. So the probability of vector religation strongly reduced (unless there is single cut plasmid present ie the digestion was not complete.)

3. Remake the insert.

Of the 3 options i am most in favour of option 2. You may have to screen more colonies than usual (if you are not confident that the digest has gone to completion), but the chances are very good that you will obtain your desired plasmid.

Hi,

Fell free to go ahead for ligation reaction.

Next time whenever you are using two different enzymes for digestion, No need to go for CIAP treatment also.

So feel free and Continue cloning work.

With regards,

Mole .

Thanks guys! That is pretty much what I figured. I might as well go ahead as I already have the digests, and I will use a large excess of insert over vector, since there is not chance of getting mutliple concatenated inserts. I just had a bad cloning day yesterday Hate it when you try to do everything so carefully but yet still manage to screw things up.