Zyppy MiniPrep Protocol
#D4020 from Zymo Research

1. For each miniprep, grow 2 ml culture, 37° C, overnight (O/N) in appropriate medium and anitbiotic (usually ampicillin,but not always); shake at 400 RPM and make sure the cultures are well aerated. You can grow as much as 8 mL of overnight culture and repeatedly pellet the cells into a microfuge tube to get more plasmid from each miniprep.
   
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2. Add 600 µL of O/N culture to an appropriately labeled 1.5 mL microfuge tube. Save the rest of the O/N culture at +4 ° C and keep them sterile. These will only survive for about 24 hours at +4 ° C.
   You can increase your yield by pelleting up to 4 mL of O/N culture and resuspending pellet very well in 600 µL water.
3. Add 100 µL 7X Lysis Buffer (blue color). Mix by inverting the tube 4-10 times. Solution should become clear instead of opaque.
   Proceed to the next step within 3 minutes.
4. Add 350 µL of Neutralization Buffer (yellow color; RNase A already added) and mix by inverting the tube until the entire solution and precipitate is yellow. This buffer is stored at +4 ° C.
5. Spin full speed for 2 minutes at RT°.
6. Prepare Zymo-Spin II column (with binding resin) by inserting into 2 mL collection tube. Be sure to label the spin column and the collection tube. Also label a 1.5 mL microfuge tube for use in step 14.
7. Transfer ~900 µL supernatant to Zymo-Spin II column. Do not transfer any of the solid precipitate.
8. Spin full speed for 15 seconds at RT°.
9. Discard liquid flowthrough and reinsert Zymo-Spin II column into same collection tube.
10. Add 200 µL Endo-Wash Buffer to the Zymo-Spin II column. Spin full speed for 15 seconds at RT°. Repeat this step a second time. No need to empty flow through. until second wash is completed.
11. Add 400 µL Zyppy Wash Buffer (with ethonol already added). Spin full speed for 30 seconds at RT°. Discard liquid flowthrough. Repeat this step a second time. Discard liquid flowthrough and and the 2 mL collection tube after the second wash. The DNA is still in the spin column.
12. Transfer Zymo-Spin II column to a clean and appropriately labeled 1.5 mL microfuge tube (from step 6 above).
13. Add 30 µL Zyppy Elution Buffer to the center of the to the Zymo-Spin II column. Let it stand for 1 minute to maximize yield.
14. Spin full speed for 30 seconds at RT°. SAVE THE LIQUID with your plasmid. Reaload the eluate onto the same column and let it sit for one minute. Spring the column again and recapture the eluate in the same tube. Discard the spin column. Store DNA at -20° C.
15. If you want to digest some DNA, 5-10 µL of the MP DNA in a final volume of 20 µL is a nice place to start. You can NanoDrop the DNA if you need to know the concentration.

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