Forgot to inactivate SAP (shrimp alkaline phosphatase) - (May/31/2007 )
Hi,
I used SAP (shrimp alkaline phosphatase) to dephosphorylate my sticky end vector and loaded it on agarose gel - forgot to deactivate it.
After gel cleaning I need it for Roche ligation.
I think i will see a band shift due to SAP attached to DNA.. on the gel..
But is it useless now for ligation or Can I still use it for Roche ligation ??
Thanks.
Gel cleaning will most likely remove proteins (as SAP). Consider the protocols that come with kits, eg. Qiagen kits, they use mostly the same buffers for PCR cleanup (enzyme removal), only the buffer to dissolve your gel is a different one. If you're not sure, just clean up from gel and then proceed with regular inactivation, then proceed with ligation.
Since SAP wasnt deactivated and was in contact with the DNA for longer than expected, some of it could run with the DNA and may damage the ends of the vector. remember there is some problem with ligation.
Good Luck !!!
once i faced this. i think the CIAP that was still present in my vector, damaged its ends badly and i tried 24 colony for miniprep, and none was my desired plasmid
I would suggest using Antartic Phosphatase from NEB. This phosphatase can be heat inactivated.
Most of the time, the presence of inactivated phosphatase will give lots of troubles to ligation reaction. I faced it before.
there is a possibility that the ends are trashed and no longer ligatable. Personally I would start again. However if you want have the curiosity, you can do a trial.. ligate your present DNA, test the resulting colonies and see what happens.
There're three ways of inactivating alkaline phosphatase, ie. heat inactivation, organic extraction (phenol-chloroform-isoamyl alcohol) and last, gel purification using a solubilization solution that contains chaotropic agent such as guanidine thiocyanate (that can denature proteins, such as alkaline phosphate).
Theoretically, there shouldn't have been a problem unless, if you had used too much CIAP during dephosphorylation step... optimal would be 0.01U/pmole ends.