Can I use my PCR product (amplified with primers having restriction sites on their ends for both enzymes) directly for restriction with (double digest) two Restrction enzymes ie. Eco RI and Nco I. Actually the buffer for the two enzymes will be used is NE Buffer 3.1 with 100% activity for Nco I and 50% activity for Eco RI. I want to know that, can both the Restriction enzymes will be compatible with PCR buffer?
Hii...
Vipasha Verma..
I think both the RE's are compatible with buffer "Cutsmart buffer (Buffer 4 + BSA". So you can use both the enzyme in a single reaction.
But I would like to suggest you that you should go with the sequential digestion...!!
Best wishes..
Hi, your PCR reaction contents should not influence your digest reaction. For the 50% activity just add double the amount of enzyme, and you can do the double digest at the same time.
Hi Vipasa!
My suggestion is that you better do ethanol precipitation of your amplified PCR product. That you can do with chloroform isoamyl (29:1) take the upper part after vigorously shaking. add sodium acetate and 100% ethanol. incubate in -20 degree overnight. next morning centrifuge at high rpm then dry the pellet after washing with 70% ethanol. I will not suggest you for double digestion because these enzymes are no High Fidelity enzymes and having 50% and 100% activity in same buffer. Better you go for sequential digestion, that will give you better results. Don't forget to do ethanol precipitation after 1st digestion.
Good Luck.
The suggestion by Fransisco Lakay is right; so it is worthy to try. Presumably, the two enzyme system can work.
Cheers
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