1. Set up the enzyme digestion as following
| |
plasmid (0.2mg/ml) | 5 μl |
10X buffer | 1 μl |
Each of restriction Enzyme (10 units/μl) | 2 μl |
ddH2O | 2 μl |
Final volume | 10 μl |
Incubate the digestions at 37°C for 1 hour.
2. Make a 160ml 1.2% agarose gel in 1X TAE with 12 μL DNA View.
(1) Measure 1.92g agarose powder to a 500ml-bottle, and add 160ml 1X TAE buffer.
(2) Melt agarose solution in microwave until the powder is dissolved. Be careful not to over-heat it, or else too much
water may evaporate and result in gels of the incorrect agarose concentration.
(3) Let gel solution cool down to 60-70°C (very warm but not hot).
(4) Add 12 μl of DNA View.
(5) Pour agarose to the gel holder with the gel comb (25-well tooth) in place. After the agarose gel is solidified,
transfer it to the gel running chamber. Fill the box with 1X TAE buffer enough to cover the whole gel.
4. Briefly centrifuge the digestion reactions and add 3 μL 6X loading dye to each sample.
5. Run digestion reactions on agarose gels in the following order:
(1) 1kb-DNA ladder, 5μl
(2) Uncut DNA control, 4 μl
(3) Digested wild-type sample, 18 μl
*Uncut DNA control: In microcentrifuge tubes add 5 μl Uncut DNA, 5 μl of ddH
2O, and 2 μl of 6X DNA loading dye.
Mix well by gentle pipetting.
6. Visualize DNA bands in agarose gels with a camera - equipped gel documentation system. Take a picture.
7. Bring agarose gel to UV light box and cut out the wanted bands. Place the gel slices into clean microcentrifuge tubes.
8. Dissolve the gel slices with 500 μl buffer for 10 min at 550℃. Invert the tubes 3 times every 3 min.
9. Take gel extraction columns and fit them into empty collection tubes. Transfer the gel/DNA solutions to columns.
10. Centrifuge columns at 13000rpm for 30 sec. Pour off all flow-through and put column back in the original collection tube.
11. Add 500 μl wash buffer (contains ethanol) to column.
12. Centrifuge at 13000rpm for 30sec.
13. Pour off all flow-through and put column back into original collection tube.
14. Centrifuge again for 2 min at 13000rpm to remove all ethanol.
15. Transfer columns to clean microcentrifuge tubes.
16. Add 30μl elution buffer to the center of the column membrane.
17. Centrifuge for 2 min at 13000rpm to elute DNA off the column.
18. Store eluted DNA in -200°C freezer .