help trouble shooting: PstI/SapI cloning - Is SapI a nasty enzyme? (Dec/10/2007 )
Thanks for all the warm discussion before.
Now I'm doing a "simple" cloning, that is to paste a PCR product with PstI/SapI ends into a similarly double-digested expression vector. However, I can never get this work. I used 1uL of each enzyme in a 10uL PCR product digestion reaction. The sticky end PCR product was gel purified before ligation. No colony has ever been obtained.
Anyone has any idea about the enzymes I used?
Are there other tricks to do the cloning?
Thank you in advance!
What is the final volume for the digestion? Better in a larger volume than small. We usually use 50ul digestion reactions. Also we gel purify the PCR product before we digest it. Some people purify through PCR purification columns and digest it.
I do not have any experience with sap1. I have used Pst1 and its works fine.
Thank you for reply!
I made the final volume as 10uL: 1uL of PstI, 1uL of SapI, 1uL of buffer, 7uL of gel purified PCR product.
When you do 50uL, how much enzyme do you use? Or you actually calculate the DNA amount?
With the reaction you indicated, you have 10% glycerol (20% of a 50% enzyme mixture), which is well above where bad things happen. Do the digestion at 50ul with the same amount of enzyme and 5 ul of the 10x buffer.
I would use 1ul of each enzyme in a 50ul reaction. We do not calculate DNA amount. We digest all of the PCR product.
Great! Thanks for all the warm replies!
Yeah...seems the glycerol concentration is too high.
When you do the 50uL digestion with 1uL of each enzyme, how long do you keep it at 37degC?
Normally we leave the digests for 2 hrs in 37C.