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Public: PlasmidMiniprep

Summary

This protocol is used to isolate plasmid from bacterial culture using the Qiagen Mini Prep Spin Kit.

Materials

• Qiagen Mini Prep Spin Kit, including spin columns
• 1.5 mL eppendorf tubes (2 per sample)
• 15 mL centrifuge tubes (1 per sample)
• 1000µL pipet and non-sterile tips
• 50µ" pipet and non-sterile tips

Equipment

• Centrifuge
• Microcentrifuge
• 70°C oven

Timeline

Day 1: Start cultures
Day 2: Isolate Plasmid

Protocol

Day 1

1. Start 1 to 5 mL of culture with relevant antibiotics. Incubate 12-16 hours overnight in 37°C shaker (adjust for plasmids with temperature sensitive replication). Use more culture for large or low copy number plasmids.

Day 2

1. Transfer culture to centrifuge tube and spin down in centrifuge for 5 minutes at 4000 rpm (around 3700g). A firm pellet should form.
2. During this time, turn on 70°C oven in PCR room and put in a tube of Milli-Q.
3. Pour off supernatent into waste beaker and invert tube on paper towel, tapping gently to make sure you get all the media off.
4. Get Buffer P1 from fridge (in plastic tub on bottom shelf). Resuspend pellet in 250µL of P1 by gently pipetting and transfer to a labeled 1.5mL epi. Be careful with the first few buffers as they are acidic/basic. If you get them on your skin, wash immediately. Once this step is finished, put P1 back in fridge. It is unusable if you leave it out at room temperature.
5. Add 250mL of Buffer P2 (found in Qiagen spin kit in PCR room) and mix by quickly and vigorously inverting the tube several times (DO NOT VORTEX). The solution should turn from cloudy to clear and there should be no precipitation. If lyseBlue had been added to P1, the solution should turn blue.
6. Add 350mL of Buffer N3 and mix by quickly inverting the tube several times. The solution should turn homogenously cloudy. If lyseBlue has been added, solution will turn clear. Keep mixing until any precipitation is gone.
7. Put epis in the microcentrifuge for 10 minutes at 15,000 rpm. Make sure load is balanced and lid is screwed on. Use this time to label your spin columns and collection epis.
8. After centrifuging is done, very carefully pull out your samples and put them in the epi rack. The pellet is very loose and you don’t want to disturb it.
9. Very carefully transfer supernatent from epi to the top of the spin column by pouring or pipetting. You don’t want to get the cell debris from the pellet onto the column.
10. Let stand on column for 1 minute. Centrifuge at 9,000 rpm for 30 seconds. Discard flow through into buffer waste bottle.
11. Optional step: If isolating a large, low copy number plasmid from a large volume of culture, you can add 500µL of buffer PB (found in PCR purification kit) to the column to wash off excess debris and centrifuge for 30 seconds at 9,000 rpm.
12. Add 750mL of Buffer PE to column. Let stand for 1-5 minutes (to eliminate extra salts) and then centrifuge for 30 seconds at 9,000 rpm and discard flow through into buffer waste bottle. Make sure ethanol has been added to the PE. If you are going to using the plasmid for cloning or other sensitive applications, it is best to use Buffer PE with 200 proof ethanol, not 190 proof, as the 190 proof often has methanol or other DNA damaging compounds added.
13. Centrifuge again at 13,000 rpm for a minute. It is important to get all ethanol out of the column as ethanol contamination will ruin all your downstream applications. Also check the inside of the column for any lingering beads.
14. Transfer the inside column to the labeled epi. Apply 30µL of 70°C Milli-Q directly to the center of the column filter and let stand for 1 minute before centrifuging for 1 minute at 13,000 rpm. Cap and you are done!

Caveats and Notes

• Plasmids are stable at room temperature for a few days but should be stored in the -20°C freezer for longer term use. They should be transformed into competent cells for permanent storage in the -80°C freezer.
• Eluting into MilliQ is better for cloning applications and sequencing, but is less stable than eluting into Tris buffer. Isolated plasmid should only be stored in the -20°C freezer for a few months, after which DNA damage can occur.
• All new plasmids should be transformed into a lab strain, given a BK number in the strain database, and stored permanently in the -80°C, so the plasmid can be extracted by everyone as needed in the future.

Original protocol from Qiagen Miniprep Handbook

Protocol Updated by: H. Lindsey Date: 12 Sept 12

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