WangLab:DNA Digestion

DNA Digestion

 * 1) make mixture of DNA digestion reaction (total of ~10 ul):
 * 2) * a.	Desired DNA(e.g. from Miniprep) 					1 ul
 * 3) * b.	10X reaction buffer (dependent on enzymes) 			1 ul
 * 4) * c.	enzyme 1 (e.g. BamHI)						0.5 ul
 * 5) * d.	enzyme 2 (e.g. EcoRI)						0.5 ul
 * 6) * e.	H2O									1 ul
 * 7) * f.	100X BSA (Optional)						0.1 ul
 * 8) incubate the mixture in 37oC incubator for 2-3 hr.
 * 9) During the waiting time, prepare 1X TAE buffer (dilute the 50X TAE stock solution with H2O).
 * 10) make 50 ml 1% agarose gel solution for 1 gel tray (i.e. 0.5 agarose in 50 ml 1X TAE buffer).
 * 11) Microwave about 40 sec-1min until boiling and agarose completely dissolved. (Caution! avoid the over-spill).
 * 12) add 2.5 ul of EB and swirl to dissolve.
 * 13) cool down a bit and pull the solution the gel tray of DNA electrophoresis system.
 * 14) Place desired combs in the gel solution to create the wells.
 * 15) 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).
 * 16) load the reaction mixture in the gel wells (Don’t forget the DNA ladder lane).