vector: Klenow + CIP - do they work together?! (Jan/30/2008 )
I have a vector which needs to be blunted and dephosphorylated before vector+insert ligation. So after I cut vector with restriction enzyme (XbaI, 5' overhang) i use Klenow to make blunt ends, but I also need to use phosphatase before ligation so I wonder can I just add phosphatase after heat inactivation of Klenow!?
this is my procedure:
- restriction: XbaI, (Fermentas, yellow buffer)
- heat inactivation of RE
- I just add Klenow, dNTPs, and a little more yellow buffer
- heat inactivation of Klenow
- then I add ~1ul CIP
- heat inactivation + EDTA
- gel extraction
- vector + insert ligation
Both klenow and CIP work in yellow Fermentas buffer so I thought it could be performed in a same mixture. But is it ok, to use Klenow and CIP like that?
How do you do this (blunting and dephosphorylating vector)?
Can excess dNTPs be a substrate for phosphatase?
You can anticipate problems with heat killing Klenow unless you add EDTA prior to the heat kill. See the Klenow pages on the NEB web site. Klenow will be active and cut back the denatured ends that occur at high temperature until it is denatured. You may also find that the CIP will need extra time, since it will be occupied by the dNTPs you have in the mix. Between the two of those, I'd add EDTA, heat kill the Klenow, precipitate the DNA, and then do the CIP treatment. Except i wouldn't use CIP, I'd use SAP or antarctic phosphatase.