Plasmid Purification

Goal

• Goal: To prepare plasmids from a strain of bacteria

Pre-Protocol Questions

1. Do you know how to properly streak a strain onto a plate using an inoculating loop?

2. Do you know how to pick a single colony from a plate?

3. Do you have liquid LB media? Sterile Erlenmeyer flasks? Milli-Q water? Virkon solution? Any other materials you will need for this protocol?

4. Do you know how to use the Plasmid Miniprep Kit? The NanoDrop Machine?

Protocol

Day 1: Streaking Plasmid Strain From Freezer Stocks

• Refer to the plasmid database (on the Microbial Evolution and Ecology Webpage) to determine which strain you plan to remove from the -80 degree Celsius freezer stock.

1. Practicing Bio safety 2 protocols, bring an ice box to the -80 degree Celsius freezer (located in S223 instrument room). Obtain desired strains and put them in the ice box.

2. Under a sterile hood, use an inoculating loop to pick the desired strain. Streak out onto pre-labeled culture plate (date, plasmid code, and plasmid name).

a. The type of culture plate is dependent on the strain type. For example, the P-10 strain is resistant to chloramphenicol. Therefore, it is streaked onto TSA + chloramphenicol plates.

3. Dispose of the contaminated inoculating loop in 5% Virkon S solution.

4. Place streaked culture plates face down in the 37 degree Celsius incubator and allow them to incubate overnight.

Day 2: Liquid Inoculation

• Practice sterile technique under the hood.

1. Get Liquid LB Media (in the cupboard under the pH meter).

2. Get sterile Erlenmeyer flasks with glass beaker lids.

3. Heat / Turn on shaking incubator to 37.3 degrees Celsius.

4. Get culture plates from 37 degree Celsius incubator.

5. Fill Erlenmeyer flasks with 10 mL of LB.

a. Refer to Working with Antibiotics to help determine the amount of antibiotic needed to add to each Erlenmeyer flask.

6. Pick a single colony from a culture plate. Put into LB media and mix. Make sure to recap the Erlenmeyer flask with the glass cap.

7. Dispose of the contaminated inoculating loop in 5% Virkon S solution.

8. Put Erlenmeyer flasks with bacteria into 37.0 degree shaking incubator overnight.

• Note: We have experimentally found that we get 5-10 times more plasmid by leaving the bacteria in liquid culture for 3 days (i.e. inoculating the liquid on a Friday and performing the plasmid prep on Monday). This seem true for both BL21 and DH5a E. coli. However, this is not a good idea if you are using ampicillin / carbenicillin.

Day 3: Plasmid Miniprep Kit — Zymo Research Miniprep Kit

There are slightly different protocols depending on how much plasmid is needed. If we need a smaller amount — testing if a plasmid is correct, for example — use procedure 'A'. If we need a large amount of the plasmid — it is a plasmid that we often use, such as P-24 or P-96 — then use procedure 'B'.

Procedure A — Smaller-scale Plasmid Purification

Before Starting:

1. Grab Ice Bucket and ice. Place ZymoPURE P3 on ice for 30 minutes before using.

2. Check if ethanol has been added to ZymoPure Wash2. If you believe it has not, then contact Eric. You can check with the checkbox on the label of Wash2 Concentrate.

Starting:

1. Obtain flasks with cloudy bacteria solution. Should be at least 6mL per flask.

2. Centrifuge 2.5mL of bacterial culture in a clear 1.5mL tube at 5,000 x g for 60 seconds in a microcentrifuge. Do this by pipetting 1.25ml (2 x 625ul on a pipettor) twice. Each time, discard the supernatant.

3. Add 250uL of P1 (Red: Kept in fridge in "Buffers" box) to the pellet and resuspend cells by pipetting and vortexing. This requires a lot of hard, full-force vortexing. It might help to vortex for 15-30 seconds, wait 5 minutes, and retry.

4. Add 250uL of P2 (Blue) and IMMEDIATELY MIX by gently inverting the tube 6-8 times. DO NOT VORTEX! Let sit at room temp for 3 mins. This addition lyses the cells, and the solution should be clear, purple, homogenous and viscous when the process is done.

5. Add 250uL of ice-cold P3 (Yellow) and mix throughout by inverting. DO NOT VORTEX. Invert the tube again 3-4 more times after the sample turns completely yellow. It will turn yellow when the neutralization is complete and a yellowish precipitate will form.

6. Incubate the neutralized lysate on ice for 5 minutes.

7. Centrifuge this neutralized lysate for 5 minutes at max speed (17,000 x g).

8. During this time, add 275ml of ZymoPURE Binding Buffer to clean, labeled 1.5mL microcentrifuge tubes.

9. Transfer 600uL of the supernatant from step 7 into the tube for step 8. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube. Vortex the 1.5 microcentrifuge tube (that contains Binding Buffer and supernatant) for 15 seconds.

10. Place a Zymo-spin II-P column in a Collection Tube; label the side of the column with the sample name. Transfer the entire mixture from step 9 into the Zymo-Spin II-P Column. One Collection Tube/Column per plasmid.

11. Incubate the Zymo-Spin II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 13,000g for 1 minute. Discard the flow-through.

12. Add 800 uL of ZymoPURE Wash 1 to the Zymo-Spin II-P Column and centrifuge at 13,000g for 1 minute. Discard the flow-through.

13. Add 800 uL of *ZymoPURE Wash 2* to the Zymo-Spin II-P Column and centrifuge at 13,000g for 1 minute. Discard the flow-though.

14. Repeat the above wash step (#15) with 200 uL of *ZymoPURE Wash 2*. Discard the flow-though

15. Centrifuge at 13,000g for 1 minute in order to remove any residual wash buffer.

16. Transfer the Zymo-Spin II-P Column into a clean (and labeled) 1.5 mL tube and add 26uL of ZymoPURE Elution Buffer directly to the column matrix. Incubate at room temperature for 2 minutes and then centrifuge at 13,000g for 1 minute in a microcentrifuge. During this spin, the lid to the microcentrifuge tube will be open.

Procedure B — Larger-scale Plasmid Purification

Before Starting:

1. Grab Ice Bucket and ice. Place ZymoPURE P3 on ice for 30 minutes before using.

2. Check if ethanol has been added to ZymoPure Wash2. If you believe it has not, then contact Eric. You can check with the checkbox on the label of Wash2 Concentrate.

Starting:

1. Obtain flasks with cloudy bacteria solution. Should be 10mL per flask. Split each flask between two 1.5mL tubes.

2. Centrifuge 5mL of bacterial culture in a clear 1.5mL tube at 5,000 x g for 60 seconds in a microcentrifuge. Do this by pipetting 1.25ml (2 x 625ul on a pipettor) four time. Each time, discard the supernatant. Gradually add volumes of culture until you get through approximately all 5mL of the culture, per tube.

3. Add 250uL of P1 (Red-should be in fridge) to the pellet and resuspend cells by pipetting and vortexing.

4. Add 250uL of P2 (Blue) and IMMEDIATELY MIX by gently inverting the tube 6-8 times. DO NOT VORTEX! Let sit at room temp for 3 mins. This addition lyses the cells, and the solution should be clear, purple, homogenous and viscous when the process is done.

5. Add 250uL of ice-cold P3 (Yellow) and mix throughout by inverting. DO NOT VORTEX. Invert the tube again 3-4 more times after the sample turns completely yellow. It will turn yellow when the neutralization is complete and a yellowish precipitate will form.

6. Incubate the neutralized lysate on ice for 5 minutes.

7. Centrifuge this neutralized lysate for 5 minutes at max speed (17,000 x g).

8. During this time, add 275ml of ZymoPURE Binding Buffer to clean, labeled 1.5mL microcentrifuge tubes.

9. Transfer 600uL of the supernatant from step 7 into the tube for step 8. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube. Vortex the 1.5 microcentrifuge tube (that contains Binding Buffer and supernatant) for 15 seconds.

10. Place a Zymo-spin II-P column in a Collection Tube; label the side of the column with the sample name. Transfer the entire mixture (875ul) from step 9 into the Zymo-Spin II-P Column. One Collection Tube/Column per plasmid.

11. Incubate the Zymo-Spin II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 13,000 x g for 1 minute. Discard the flow-through.

12. Add in the supernatant + binding buffer from the same plasmid into the respective column from the previous step. So, we are putting supernatant from the same 10ml liquid culture into the same column.

13. Incubate the Zymo-Spin II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 13,000 x g for 1 minute. Discard the flow-through.

14. Add 800 uL of ZymoPURE Wash 1 to the Zymo-Spin II-P Column and centrifuge at 13,000 x g for the 1 min. Discard the flow-through.

15. Add 800 uL of *ZymoPURE Wash 2* to the Zymo-Spin II-P Column and centrifuge at 13,000 x g for 1 min. Discard the flow-though.

16. Repeat the above wash step (#15) with 200 uL of *ZymoPURE Wash 2*. Discard the flow-though

17. Centrifuge at 13,000g for 1 minute in order to remove any residual wash buffer.

18. Transfer the Zymo-Spin II-P Column into a clean (and labeled) 1.5 mL tube and add 26uL of ZymoPURE Elution Buffer directly to the column matrix. Incubate at room temperature for 2 minutes and then centrifuge at 13,000g for 1 minute in a microcentrifuge. During this spin, the lid to the microcentrifuge tube will be open.