Team:Wisconsin-Madison/notebookprotocols

1. Pellet the overnight culture(s) in a 1.5 ml or 2ml eppendorf tube. (I usually do 10,000 rpm, 3 minutes) 1 minute works fine. I usually use 3 ml culture per prep.
2. Resuspend each pellet in 200 μl Alkaline Lysis Sol I, RnaseA added (final RNase A concentration should be 100 μg/mL). Make sure there are no lumps.
3. Add 400 μl Alkaline Lysis Sol II. Invert 4-6 times to mix. Do not allow reaction to lyse for more than 5 min. Sample should clarify.
4. Add 300 μl Alkaline Lysis Sol III. Invert 4-6 times to mix. Sample should have a white precipitate.
5. Add 100 μl chloroform. Do this in a fume hood. Invert 4-6 times to mix.
6. Rest on ice for 5-10 minutes. This step is so that the chloroform does not get too hot in the centrifuges and leak out of the tubes. If you want to skip this step you might consider using less chloroform. I put the tubes at -20 for a couple of minutes.
7. Centrifuge at max. speed (14,000 rpm) for 10 minutes.
8. Pipet 750μl of supernatant/aqueous layer into a fresh tube. I do up to 800 uL
9. Add 1/10 volume (75μl) 3M NaOAc, pH 5.2. Vortex/flick to mix. 80 uL
10. Add 0.7-1.0 Volume COLD isopropanol. Vortex/flick to mix. If in a hurry go straight to step 11, otherwise rest on ice for 10-30 minutes. I have even let it precipitate overnight at 4°C if convenient. 600 uL isopropanol. Then I put it at -20 for 5 minutes up to over the weekend if needed.
11. Centrifuge at max. speed for 25 min. Most miniprep protocols say to do this at 4°C, but I have not noticed decreased yield by centrifuging at room temp.
12. Remove and discard the supernatant. Don’t disturb the pellet. Sometimes I can’t see a pellet, and more often than not I still have DNA.
13. Add 1ml of 70% EtOH (at room temp.). Invert 4-6 times to rinse the tube.
14. Centrifuge at max speed for ~5 minutes. Room temp. is fine. Remove and discard the EtOH.
15. Repeat steps 14 and 15 to remove all traces of isopropanol. Pulse spin after removing bulk of final EtOH wash and pipet off remaining EtOH.
16. Air dry the pellet for ~15 minutes (pellet will change from white to clear as it dries). Resuspend in desired volume of H2O or T10E1, depending on downstream applications. If you pipet off the EtOH well, then I have done this for as little as 2 minutes before. For fosmids, I usually resuspend the pellet in 20 uL water.

Digestion

For screening, you only need a small amount to run on a gel - 10uL rxn

Check enzyme compatibility, what buffer is needed, and whether BSA is necessary

• DNA - 2uL (usually fine)
• Buffer(10x) - 1uL
• BSA(10x) - 1uL
• Enzyme - 0.4uL each (ADD LAST and no more than 10% of rxn volume)
• Water - fill to 10uL

1-2 hours in 37C waterbath (check NEB if you want quicker time)

• Add 2uL of 6x Dye
• Load 6uL in gel

During cloning, you will need to digest more DNA for gel extraction - 50uL rxn

Check enzyme compatibility, what buffer is needed, and whether BSA is necessary

• DNA - 2ug
• Buffer(10x) - 5uL
• BSA(100x) - 0.5uL
• Enzyme - 2uL each (ADD LAST and no more than 10% of rxn volume)
• Water - fill to 50uL

1-2 hours in 37C waterbath (check NEB if you want quicker time)

• Add 10uL of 6x Dye
• Load 60uL in gel

Template Destruction - 50uL rxn

Check enzyme compatibility, what buffer is needed, and whether BSA is necessary

If your product for digestion came directly from PCR you can destroy the original template by preforming a DpnI digestion. DpnI will digest methylated DNA. PCR product is unmethylated. If needed, do this step before cloning digestion.

• 1uL DpnI/50uL rxn
• incubate in 37C waterbath for 1 hour