Midiprep R334C

1. Transfer cultures into 14 mL Falcon tubes and spin at 5,000 g for 3 minutes. Discard supernatant into waste jar.

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

Might need to add more

3. Add about 1 mL of 3 M sodium acetate pH 5.2. Vortex for 10 seconds.

4. Centrifuge for 3 minutes at 5,000 g and transfer supernatant to new, previously labeled Falcon tube.

5. Add 2.5 µL of RNase (Enzyme Rack, Freezer A) and wait for 5 minutes.

6. Add about 1 mL of PCl (Fridge A) from the bottom layer. Vortex for 10 seconds and centrifuge at 5,000 g for 5 minutes.

7. Transfer the top layer to a new, previously labeled Falcon tube. Add about 1 mL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 5,000 g for 3 minutes.

8. Transfer the top layer to a new, previously labeled Falcon tube. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 5,000 g for 5 minutes. Proceed to sodium acetate precipitation.

D. Ethanol Precipitation of Dilute DNA

1. 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.

2. Estimate the new volume of the DNA sample. Add 2 times the estimated volume (added 40uL) in 100% ethanol (Fridge A), and spin at 13,000 rpm for 5 minutes.

3. 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.

4. Carefully decant supernatant down the drain following the same procedure as step 3. Circle where the DNA on the tube is. Leave the tubes uncapped and place a Kimwipe over the top.

5. Re-suspend the dry DNA in 20 µL of MBG water (Fridge A), vortex, and transfer to a previously labeled 1.5 mL tube.

Labeled R334C ET III