Miniprep R334C

1. Transfer 2 mL of the bacterial culture into 2 mL tubes and spin at 13,000 rpm for 1 minute. Discard supernatant into waste jar, leaving about 50-100 µL in the tube. Repeat this step if there are 4 mL of bacterial culture.

2. Add 300 µL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

3. Add 150 µL of 3 M sodium acetate buffer pH 5.2. Vortex for 10 seconds.

4. Centrifuge for 5 minutes at 13,000 rpm and transfer supernatant to new, previously labeled 1.5 mL tube.

5. Add 1.5 µL of RNAse (Enzyme Rack, Freezer A) and wait for 5 minutes.

6. Add 450 µL of PCl (Fridge A) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 5 minutes. Use PCI under the fume hood. Dispose of pipet tip in chemical tip waste bin (located in fume hood).

7. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 450 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 rpm for 5 minutes.

8. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 13,000 rpm for 5 minutes.

9. Estimate the volume of the DNA sample. Add 1/10 of the estimated volume in 3 M sodium acetate buffer pH 5.2 (Cabinet A) and mix by inverting 10 times. Leave overnight.

10. Estimate the new volume of the DNA sample. Add 2 times the estimated volume in 100% ethanol (Fridge A), and spin at 13,000 rpm for 5 minutes (10uL 100% ethanol added).

11. Carefully decant supernatant down the drain by turning the tube(s) until completely upside down. Touch the tube to a Kimwipe to wick away any residual ethanol. Add 1 mL 70% ethanol (Fridge A) and centrifuge at 13,000 rpm for 1 minute.

12. Carefully decant supernatant down the drain following the same procedure as step 6. Leave the tubes uncapped and place a Kimwipe over the top. Leave overnight.