Linearization of MiniPrep Plasmid DNA

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1. Make Linearization Master Mix:

per reaction:

10 uL 10 X NEB Buffer #3

1 uL BSA 100 X

61 uL DI H2O

3 uL NEB Not1 (10 U / uL)

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75 uL

 

2. In a sterile 1.5 mL microcentrifuge tube, add 75 uL of Linearization Master Mix to 25 uL of mini prep plasmid DNA (5 10 ug). Mix by pipetting.

 

3. Incubate at 37C overnight (> 14 hr).

 

4. Run a few representative samples on a 1 % agarose gel (ex: 0.5 g agarose + 1 mL 50 X TAE buffer + ~49 mL DI H2O) to verify linearization. (each sample 1 uL reaction + 9 uL DI H2O + 1.7 uL 10 X Sample buffer) (Run at 170 V for 5 minutes against a 1 kb ladder)

 

5. Add 100 uL of phenol / chloroform / isoamyl alcohol (25:24:1) to each tube. Vortex for 30 seconds and then spin at high speed for 5 minutes. Transfer 95 uL of the aqueous phase (top layer) into a sterile 1.5 mL tube. Make sure no interface or organic phase is removed.

 

6. Add 100 uL of chloroform / isoamyl alcohol (24:1) to each tube. Vortex for 30 seconds and then spin at high speed for 5 minutes. Transfer 95 uL of the aqueous phase (top layer) into a sterile 1.5 mL tube. Make sure no interface or organic phase is removed.

 

7. Add 30 uL of 7.5 M NH4OAc (Ammonium acetate) to aqueous layer.

 

8. Add 130 uL of cold (4C) 100 % isopropanol. Vortex and place at -20C for > 30 min.

 

9. Spin tubes at high speed (4C) for > 30 min. Aspirate the supernatant with a 27 gauge needle, being careful not to aspirate the DNA pellet.

 

10. Gently add 500 uL of cold (-20C) 80 % ethanol to DNA pellet. Spin for 10 min. at 4C. Aspirate the supernatant with a 27 gauge needle.

 

11. Dry in a 37 C heat block for 15 20 min. Verify that all the EtOH has evaporated.

 

12. Resuspend dried pellet in 10 uL of sterile RNase free DI H2O and store at -20C.