Plasmid Purification: Difference between revisions

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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).  
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.  
: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 contaminated inoculating loop in 5% Virkon S solution.
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.
4. Place streaked culture plates face down in the 37 degree Celsius incubator and allow them to incubate overnight.
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5. Fill Erlenmeyer flasks with 10 mL of LB.  
5. Fill Erlenmeyer flasks with 10 mL of LB.  
a. Refer to the ''Working With Antibiotics" page on the Lab Wiki to help determine the amount of antibiotic needed to add to each Erlenmeyer flask.  
:a. Refer to [[Working with Antibiotics]] to help determine the amount of antibiotic needed to add to each Erlenmeyer flask.  


6. Pick single colony from culture plate. Put into LB media and mix. Make sure to recap the Erlenmeyer flask with the glass cap.  
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 contaminated inoculating loop in 5% virkon S solution.  
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.
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.
*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.


'''Day 3: Plasmid Miniprep Kit'''  
'''Day 3: Plasmid Miniprep Kit'''  


Before Starting:  
''Before Starting:''


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


2. Prepare Buffer:
2. Prepare Buffer:
:a. Make 95% EtOH. For 50mL, add 47.5mL 100% EtOH to 2.5mL Mili-Q H2O.  
:a. Make 95% EtOH. For 50mL, add 47.5mL 100% EtOH to 2.5mL Mili-Q H2O.  
:b. Add 42mL of this to 11mL of ZymoPure Wash2 Concentrate (D4209).
:b. Add 42mL of this to 11mL of ZymoPure Wash2 Concentrate (D4209).
3. The ZymoPURE P2 and ZumoPURE Binding Buffer may have precipitated.  
3. The ZymoPURE P2 and ZumoPURE Binding Buffer may have precipitated.  
a. IF SO-dissolve the precipitate by incubating the bottles at 30-37 degrees C for 10-20 minutes and mix by inversion. DON’T microwave.  
:a. If so, dissolve the precipitate by incubating the bottles at 30-37 degrees C for 10-20 minutes and mix by inversion. DON’T microwave.
 
''Starting: (Perform everything at room temperature (15-30 degrees C).''
 
4. Obtain flasks with cloudy bacteria solution. Should be 10mL per antibiotic. Split each flask between two 1.5mL tubes.  


Starting: (Perform everything at room temperature (15-30 degrees C).
5. Centrifuge 5mL of bacterial culture in a clear 1.5mL tube at full speed for 20 seconds in a microcentrifuge. Discard the supernatant. Gradually add volumes of culture until you get through 5mL of the culture, per tube.  
Obtain flasks with cloudy bacteria solution. Should be 10mL per antibiotic. Split each flask between two 1.5mL tubes.
:a. Centrifuge 5mL of bacterial culture in a clear 1.5mL tube at full speed for 20 seconds in a microcentrifuge. Discard the supernatant. Gradually add volume of culture until you get through 5mL of the culture, per tube.  


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


5. Add 250uL of P2 (Green) 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.
7. Add 250uL of P2 (Green) 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.
   
   
6. 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.  
8. 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.  


7. Incubate the neutralized lysate on ice for 5 minutes.  
9. Incubate the neutralized lysate on ice for 5 minutes.  


8. Centrifuge this neutralized lysate for 5 minutes at 16,000 x g.
10. Centrifuge this neutralized lysate for 5 minutes at 16,000 x g.


9. Transfer 600uL of the supernatant from step 6 into a clean 1.5mL microcentrifuge tube. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube.  
11. Transfer 600uL of the supernatant from step 6 into a clean 1.5mL microcentrifuge tube. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube.  


10. Add 275 uL of ZymoPURE Binding Buffer to the cleared lysate from step 7 and mix thoroughly by inverting the capped tube 8 times.  
12. Add 275 uL of ZymoPURE Binding Buffer to the cleared lysate from step 7 and mix thoroughly by inverting the capped tube 8 times.  


11. Place a Zymo-spin II-P column in a Collection Tube and transfer the entire mixture from step 8 into the Zymo-Spin II-P Column.  
13. Place a Zymo-spin II-P column in a Collection Tube and transfer the entire mixture from step 8 into the Zymo-Spin II-P Column.  


12. Incubate the Zymo-Spin II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 5,000 x g for 1 minute Discard the flow through
14. Incubate the Zymo-Spin II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 5,000 x g for 1 minute. Discard the flow-through


13. Add 800 uL of ZymoPURE Wash 1 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for the 1 min. Discard the flow through.  
15. Add 800 uL of ZymoPURE Wash 1 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for the 1 min. Discard the flow-through.  


14. Add 800 uL of ZymoPURE Wash2 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for 1 min. Discard the flow though. Repeat this wash step with 200 uL of ZymoPURE Wash 2.  
16. Add 800 uL of ZymoPURE Wash2 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for 1 min. Discard the flow-though. Repeat this wash step with 200 uL of ZymoPURE Wash 2.  


15. Place the Zymo-Spin II-P Column in a Collection Tube and transfer to a microcentrifuge. Centrifuge at > or at 10,000 x g for 1 minute in order to remove any residual wash buffer.  
17. Place the Zymo-Spin II-P Column in a Collection Tube and transfer to a microcentrifuge. Centrifuge at > or at 10,000 x g for 1 minute in order to remove any residual wash buffer.  


16. Transfer the Zymo-Spin II-P Column into a clean 1.5 mL tube and add 25uL of ZymoPURE Elution Buffer directly to column matrix. Incubate at room temperature for 2 minutes and then centrifuge at over or equal to 10,000 x g for 1 minute in a microcentrifuge.
18. Transfer the Zymo-Spin II-P Column into a clean 1.5 mL tube and add 25uL of ZymoPURE Elution Buffer directly to the column matrix. Incubate at room temperature for 2 minutes and then centrifuge at over or equal to 10,000 x g for 1 minute in a microcentrifuge.




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2. Choose the nucleotide program.  
2. Choose the nucleotide program.  


3. Wait for the software to perform routine verification test.
3. Wait for the software to perform a routine verification test.


4. Choose the appropriate extrinsic coefficient from the toggle/ "type" menu (dDNA, sDNA, oligo, etc.)
4. Choose the appropriate extrinsic coefficient from the toggle/ "type" menu (dDNA, sDNA, oligo, etc.)


5. Raise arm and clean pedistal (tap & bottom) with water.  
5. Raise arm and clean pedestal (tap & bottom) with water.  


6. Pipette 1uL of Elution Buffer onto the NanoDrop sensor. ''Make sure the elution buffer you are using is the same one used during the plasmid purification process''.  
6. Pipette 1uL of Elution Buffer onto the NanoDrop sensor.
:a.''Make sure the elution buffer you are using is the same one used during the plasmid purification process''.  


7. Close the console and press the blank button.
7. Close the console and press the blank button.
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'''Clean-up'''
'''Clean-up'''


1. Make sure to place any test tubes, inoculating loops, microcentrifuge tubes, etc. into virkon solution.
1. Make sure to place any test tubes, inoculating loops, microcentrifuge tubes, etc. into Virkon solution.


2. Throw out any other left-over materials.
2. Throw out any other left-over materials.


3. Clean the balances and any other possibly dirty surface.
3. Clean the balances and any other possibly dirty surfaces.

Revision as of 12:59, 1 March 2021

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.

Day 3: Plasmid Miniprep Kit

Before Starting:

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

2. Prepare Buffer:

a. Make 95% EtOH. For 50mL, add 47.5mL 100% EtOH to 2.5mL Mili-Q H2O.
b. Add 42mL of this to 11mL of ZymoPure Wash2 Concentrate (D4209).

3. The ZymoPURE P2 and ZumoPURE Binding Buffer may have precipitated.

a. If so, dissolve the precipitate by incubating the bottles at 30-37 degrees C for 10-20 minutes and mix by inversion. DON’T microwave.

Starting: (Perform everything at room temperature (15-30 degrees C).

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

5. Centrifuge 5mL of bacterial culture in a clear 1.5mL tube at full speed for 20 seconds in a microcentrifuge. Discard the supernatant. Gradually add volumes of culture until you get through 5mL of the culture, per tube.

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

7. Add 250uL of P2 (Green) 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.

8. 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.

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

10. Centrifuge this neutralized lysate for 5 minutes at 16,000 x g.

11. Transfer 600uL of the supernatant from step 6 into a clean 1.5mL microcentrifuge tube. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube.

12. Add 275 uL of ZymoPURE Binding Buffer to the cleared lysate from step 7 and mix thoroughly by inverting the capped tube 8 times.

13. Place a Zymo-spin II-P column in a Collection Tube and transfer the entire mixture from step 8 into the Zymo-Spin II-P Column.

14. Incubate the Zymo-Spin II-P/Collection Tube assembly at room temperature for 2 minutes and then centrifuge at 5,000 x g for 1 minute. Discard the flow-through

15. Add 800 uL of ZymoPURE Wash 1 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for the 1 min. Discard the flow-through.

16. Add 800 uL of ZymoPURE Wash2 to the Zymo-Spin II-P Column and centrifuge at 5,000 x g for 1 min. Discard the flow-though. Repeat this wash step with 200 uL of ZymoPURE Wash 2.

17. Place the Zymo-Spin II-P Column in a Collection Tube and transfer to a microcentrifuge. Centrifuge at > or at 10,000 x g for 1 minute in order to remove any residual wash buffer.

18. Transfer the Zymo-Spin II-P Column into a clean 1.5 mL tube and add 25uL of ZymoPURE Elution Buffer directly to the column matrix. Incubate at room temperature for 2 minutes and then centrifuge at over or equal to 10,000 x g for 1 minute in a microcentrifuge.


Measuring [DNA] via NanoDrop

  • NanoDrop Machine located in the BioSuper Lab Instrument Room (E104). Practice BioSafety 2 protocols when transferring materials outside of the lab.

1. Open the NanoDrop Software (NanoDrop 2000).

2. Choose the nucleotide program.

3. Wait for the software to perform a routine verification test.

4. Choose the appropriate extrinsic coefficient from the toggle/ "type" menu (dDNA, sDNA, oligo, etc.)

5. Raise arm and clean pedestal (tap & bottom) with water.

6. Pipette 1uL of Elution Buffer onto the NanoDrop sensor.

a.Make sure the elution buffer you are using is the same one used during the plasmid purification process.

7. Close the console and press the blank button.

8. Clean sensor with KimWipe and water.

9. Label sample then load 1 uL of sample onto the NanoDrop sensor.

10. Press the Measure button (Measures [DNA] in ng/uL)

11. Repeat steps 7-10 for each sample.

Clean-up

1. Make sure to place any test tubes, inoculating loops, microcentrifuge tubes, etc. into Virkon solution.

2. Throw out any other left-over materials.

3. Clean the balances and any other possibly dirty surfaces.

Retrieved from "http://microbes.sites.haverford.edu/MEE_wiki/index.php?title=Plasmid_Purification&oldid=2403"