Restriction digestion
For cloning:
Recipe:
Plasmid : x ul (1ug plasmid is usually good)
10XNEB Buffer : 5 ul
Enzyme 1 : 1 ul
(Optional enzyme 2 : 1 ul)
ddH2O : make up for a 50 ul
Total volume : 50 ul
Note: For HF (high fidelity) enzymes, use 10xcutsmart buffer. For none-HF enzymes, check the NEB website for buffer choice of single or double digestion. (https://nebcloner.neb.com/#!/redigest)
Incubate at 37°C overnight (I prefer overnight to get a compete digestion, but if you are pressed on time, you can also do a 2 hr digestion)
Optional: Antarctic phosphatase treatment
The purpose is to remove the 5’ phosphate of the restriction digested plasmid so that the linearized plasmid cannot re-ligate, forming empty vectors that interferes with cloning. If you used a single restriction enzyme, then this step is required because it is much easier for a single digested plasmid to re-ligate due to the matching sticky ends.
Recipe:
Restriction digested plasmid : 50 ul
10x Antarctic phosphatase buffer : 5.5 ul
Antarctic phosphatase : 1 ul
Incubate at 37°C 15 min, then incubate the reaction at 65°C for 10 min to inactivate the phosphatase. Purify the cut vector using the PCR purification kit.
For checking plasmid insert after mini prep:
Recipe:
Plasmid : x ul (1ug plasmid is usually good)
10XNEB Buffer : 2 ul
Enzyme 1 : 0.5 ul
Enzyme 2 : 0.5 ul
ddH2O : make up for a 20 ul
Total volume : 20 ul
Incubate at 37°C for 1-2 hours. Then add DNA loading dye and load the entire reaction on to the agarose gel.
Zhen
12/19/17