Plasmid for sequencing.doc 2-26-04 WSG

from Sambrook and Russel, Molecular Cloning vol 1, 3^rd ed.

1) Inoculate a single colony into 3 mL TB medium supplemented with the appropriate antibiotic to select for plasmid maintenance. Grow with aeration at 37 C for 16 to 20 hours.

2) Transfer 1.5 mL of the overnight culture to a 1.5 mL tube and centrifuge at full speed for 1 min.

3) Immediately pour off the supernatant into a container for recombinant waste. Remove the last bit of supernatant with a yellow pipet tip. Excess supernatant can interfere with subsequent steps.

4) Resuspend the pellet in 100 uL solution I by vortexing.

5) Add 200 uL of freshly prepared lysis solution and mix thoroughly by inverting the tube. Store on ice.

6) Add 150 uL of cold 3M potassium acetate, mix the tube by inversion, and store on ice for 3-5 min.

7) Centrifuge for 5 min. at full speed and decant the supernatant to a new tube.

8) Add 0.9 mL of 95% ethanol and invert several times. Leave at room T until a precipitate is visible (usually within 2 minutes).

9) Centrifuge for 5 min. at full speed. Pour off the supernatant and invert the tube with the lid open on a paper towel.

10) Add 1 mL of 70% ethanol and dislodge the pellet by finger-flicking. If finger-flicking doesn't dislodge the pellet, scrape the pellet from the tube with a yellow pipet tip.

11) Spin for 2 min at full speed (21000 x g). Pour off the supernatant and invert the tube with the lid open on a paper towel.

12) Dry the pellet completely in a speed vac, then resuspend the pellet in 104 uL RNAse solution. Incubate at 37 C for 30-60 min. to digest the RNA. Be sure that the pellet is dissolved before timing the incubation.

13) Add 104 uL chloroform and vortex briefly or (if isolating cosmid DNA) mix on a labquake for at least 5 min.

14) Spin for 5 min at full speed (21000 x g) and remove the upper phase (with a yellow pipet tip) to a new tube. Avoid the interface like the plague.

15) Add 10 uL of 3 M sodium acetate (autoclaved and stored at room T) and 220 uL 95% ethanol, then mix by inversion. Precipitate at

-20 C for 15 min.

16) Spin for 5 min at full speed (21000 x g). Pour off the supernatant and invert the tube with the lid open on a paper towel.

17) Add 0.5 mL of 70% ethanol and dislodge the pellet by finger-flicking. If finger-flicking doesn't dislodge the pellet, scrape the pellet from the tube with a yellow pipet tip.

18) Spin for 5 min at full speed (21000 x g). Pour off the supernatant and invert the tube with the lid open on a paper towel.

19) Dry the pellet completely in a speed vac, then resuspend the pellet in 100 uL of water by finger flicking.

20) Run 2 uL of plasmid DNA on a gel with a known quantity of size standard or other reference DNA. Verify that the plasmid DNA is not degraded. Estimate the DNA concentration by comparing fluorescence intensity of plasmid DNA to the fluorescence intensity of the reference DNA.

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Solution I: TEG (50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA) autoclaved then stored at 4 C.

Lysis solution: for 1 mL, mix 880 uL water, 100 uL 10% SDS, 20 uL 10 N NaOH. Make sure that no precipitate is visible. This can be stored at 37 C until used.

RNAse solution: mix 100 uL of TE and 4 uL of RNAse A (from 10 mg/mL boiled stock) for a final RNAse concentration of 400 ug/mL.

3M potassium acetate: mix 60 mL of 5M potassium acetate with 11.5 mL of glacial acetic acid and 28.5 mL of water, autoclaved then stored at 4 C.