LINEARIZING PLASMIDS
Recipe:
10l DNA (boxes 41, 42, 43) (1 g/μl)
10 μl Enzyme (the volume depends on the units of enzyme but no more of 10% enzyme
of total volume)
10 μl Buffer (box 38) (the buffer is 10X so always dilute 1:10)
70 μl ddH2O
Total volume= 100 μl
 Digest at 37 degrees C overnight
Next day:
1. Add 400 μl ddH2O to bring volume to 500 μl, shake to resuspend
2. Add 500 μl Phenol-Chloroform-Isoamyl alcohol (PCI 24: 24: 1 mL ratio); or 500 l
equilibrated phenol
3. Vortex well, spin for 3 minutes at ~13,000 rpm, transfer top layer to fresh tube
4. Add 500 μl PCI to supernatant or 250 l equilibrated phenol + 250 l chloroform; vortex,
spin again, transfer supernatant
5. Add 500 μl Chloroform to supernatant, vortex, spin, transfer supernatant
6. Add 50 μl 3M Na Acetate, then fill with isopropanol
7. Put on dry ice for approx. 10-15 minutes or leave overnight at -20°C
8. Spin at high speed in refrigerated centrifuge for 15 minutes
9. Pour off isopropanol
10. Add 70% ethanol, spin for 5 minutes
11. Pour off ethanol, speed-vac for ~5-10 minutes
12. Resuspend pellet in 10 μl ddH2O
13. prepare a 1% agarose gel
14. Using 2 μl Orange G loading dye, load 1 μl (with 7 μl dH2O) of probe to check linearization.
Run with 1 l of uncut plasmid and with 3 μl of standard (BstEII digest) to check size.
Updated 14/12/15 by LM