- make mixture of DNA digestion reaction (total of ~10 ul):
- a. Desired DNA(e.g. from Miniprep) 1 ul
- b. 10X reaction buffer (dependent on enzymes) 1 ul
- c. enzyme 1 (e.g. BamHI) 0.5 ul
- d. enzyme 2 (e.g. EcoRI) 0.5 ul
- e. H2O 1 ul
- f. 100X BSA (Optional) 0.1 ul
- incubate the mixture in 37oC incubator for 2-3 hr.
- During the waiting time, prepare 1X TAE buffer (dilute the 50X TAE stock solution with H2O).
- make 50 ml 1% agarose gel solution for 1 gel tray (i.e. 0.5 agarose in 50 ml 1X TAE buffer).
- Microwave about 40 sec-1min until boiling and agarose completely dissolved. (Caution! avoid the over-spill).
- add 2.5 ul of EB and swirl to dissolve.
- cool down a bit and pull the solution the gel tray of DNA electrophoresis system.
- Place desired combs in the gel solution to create the wells.
- when the digestion reaction is ready, take out reaction mixture, add 6X DNA loading buffer into the mixture and dilute to 1X (e.g. add 2 ul of 6X loading buffer to 10 ul reaction mixture).
- load the reaction mixture in the gel wells (Don’t forget the DNA ladder lane).