BIO430 Plasmid Purification and Analysis Laboratory Exercise
Overview
In this stage, you will analyze plasmids that have been successfully propagated to verify that they have the PCR product inserted. Purifying the plasmid from a small culture of transformed bacteria (a miniprep) produces a sufficient amount of plasmid DNA. Next, the plasmid is analyzed by restriction enzyme digestion to assess the size of the fragment inserted and to compare it to the size of the PCR product ligated into the plasmid. The blunted PCR product was inserted into the plasmid vector pJet1.2 (see figure below). pJet1.2 contains a Bgl II restriction enzyme recognition site on either side of the insertion site. Thus, once the plasmid DNA has been isolated, digestion with this restriction enzyme will allow determination of the size of any fragment inserted into the vector.
Transformed bacteria containing plasmids should be grown to late growth phase in LB
Amp broth prior to this lab.
Safety Note — The lysis solution contains sodium hydroxide, which causes burns.
Handle with care and follow standard laboratory practices, including wearing gloves and avoid contact with eyes, skin, and clothing.
Experimental Procedure for Plasmid Purification
1. Label one capless collection tube, one plasmid mini column, and two microcentrifuge tubes for each miniprep culture with your initials and the information on the labels of the
15 ml overnight cultures. On the microcentrifuge tubes, include "miniprep DNA" in the name.
2. Place each column in the appropriate capless collection tube.
3. Transfer 1.5 ml of each miniprep culture into one of the appropriately labeled microcentrifuge tubes by pipetting or decanting.
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4. Centrifuge the microcentrifuge tubes for 1 minute at top speed (>12,000 x g) to pellet the bacteria. Make sure that the microcentrifuge is balanced, and accommodate tubes of classmates to ensure economic use of the microcentrifuge.
This step pellets the bacteria.
5. Locate the bacterial pellet and remove the supernatant from each tube using a 1,000 μl pipet, avoiding the pellet. The supernatant can be added back to the original 15 ml culture and should then be disposed of by placing in the biohazard waste container.
6. Resuspend bacterial pellet in each tube in 250 μl of resuspension solution by pipetting up and down or vortexing. Use a fresh tip each time. Ensure that no clumps of bacteria remain.
7. Pipet 250 μl of lysis solution into each tube and mix by gently inverting 6–8 times.
Do not pipet or vortex this lysate or you risk shearing the bacterial gDNA, which could contaminate your plasmid preparation!
This step chemically breaks the cells open.
8. Within 5 minutes of adding lysis solution, pipet 350 μl of neutralization solution into each tube and mix by gently inverting 6–8 times.
Do not pipet or vortex this lysate. A precipitate should form.
This step precipitates out the gDNA, proteins and cellular debris.
9. Centrifuge the tubes for 5 minutes at top speed in the microcentrifuge. Make sure the microcentrifuge is balanced.
This step purifies the plasmid DNA from the bacterial lysate.
10. Decant or pipet supernatant from the centrifuged tubes onto the appropriately labeled column. Avoid transferring any precipitate. If necessary, recentrifuge the microcentrifuge tubes. Discard microcentrifuge tube.
This step binds plasmid DNA to column matrix.
11. Centrifuge the columns, still in the capless collection tubes, in the microcentrifuge for
1 minute at top speed.
12. Discard the flow-though from the collection tube and replace the column in the collection tube.
13. Pipet 750 μl of wash solution onto each column.
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14. Centrifuge columns in the capless collection tubes in the microcentrifuge for 1 minute at top speed.
15. Discard the flow-though from the collection tube.
16. Replace columns into collection tubes and centrifuge for an additional 1 minute to dry out the column. It is vital that this step be performed, or wash solution and ethanol may contaminate the purified plasmid.
17. Transfer each column to the appropriately labeled capped "miniprep DNA" microcentrifuge tube and pipet 100 μl of elution solution onto the column.
18. Let the elution solution be absorbed into the column for 1–2 minutes.
19. Place the column in the microcentrifuge tube into the centrifuge. It is best to orient the cap of the microcentrifuge tube downwards, towards the center of the rotor, to minimize friction and damage to the cap during centrifugation.
20. Centrifuge the columns for 2 minutes.
This step elutes the purified plasmid DNA.
21. Discard the columns and cap the tubes containing the eluted sample.
22. Store miniprep plasmid DNA at 4°C short-term (up to 1 month) or –20°C long-term but first, continue on with restriction digest (below).
Experimental Procedure for Restriction Digestion Analysis
23. Label one microcentrifuge tube with your initials and "Bgl II master mix".
24. Prepare a 2x master mix for Bgl II restriction digestion reactions according to the following table, using stock reagents from the common workstation. Use a fresh tip for each reagent.
* Come to Dr. Miranda to get the enzyme.
25. Label a microcentrifuge tube for each plasmid miniprep.
26. Prepare digestion reactions by combining 10 μl of the Bgl II master mix and 10 μl of each plasmid DNA in the appropriately labeled microcentrifuge tubes.
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27. Mix tube components and spin briefly in a microcentrifuge to collect the contents at the bottom of the tube.
28. Incubate reactions at 37°C for 1 hour.
29. Store the reactions at –20°C since they will not be analyzed by gel electrophoresis immediately.
30. Prepare a 1% agarose gel with 1X TBE (do not forget to add the SYBR green stain).
Be sure to use the comb that will result in more than 10 wells. Once it has solidified, wrap it in Saran wrap and store it in the refrigerator until the next lab meeting.
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