Dear All,
This is going to be a very silly question to ask. I have digested my vector(~4000bp) with ApaI and I need to dephosphorylate to prevent self ligation. I looked at the NEB protocol for both CIP& SIP alkaline phosphatase. It says I should take 1 pmole of DNA. Should I add alkaline phosphatase directly to the restriction digestion mixture that I previously used for ApaI digestion? Both are Cut Smart compatible. 1 pmoles of my Vector is equivalent to 2.8 ug.If I need to Gel purify that digested Vector, it never gets too high concentration in terms of recovery.I have never used single restriction enzyme based cloning ,but this time I have no other option
..." Should I add alkaline phosphatase directly to the restriction digestion mixture that I previously used for ApaI digestion? "...
Yes, you should. The phosphatase alkaline works very well in restriction digestion mixture.
Juan Gabriel Costa Thanks for your answer. What is the probability of having self ligated PCR product? let me explain it further. I PCRed my gene of interest and ligate them to p-GEMT vector. PCR fragment is flanked by ApaI site. Do I also need to Treat ApaI flanked PCR product with Alkaline phosphatase? Size of PCR fragments varies from 890-1700.
..."What is the probability of having self ligated PCR product?"... Does not matter. If it binds, when entering a cell, your insert has no DNA polymerase binding site. Therefore, it can not spread in cell division.
..."Do I also need to Treat ApaI flanked PCR product with Alkaline phosphatase?"... Do not treat the fragment with the phosphatase because otherwise it will not be able to bind to the vector.
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