— Christine Robbins 2018/05/01 13:00

— edited: — Dan Felker 2018/11/05 13:21

We use Qiagen's Midi Prep kit in order to extract plasmids out of bacterial cells. Please remember to plan ahead and make sure we have everything you will need to complete the protocol. This protocol also takes between 3 and 5 hours and there is only one opportunity to stop towards the end of the protocol if you are running out of time.

As with all protocols, if you feel anything in here needs more explanation to be clear, please consult with your tech and then add the appropriate details.

This protocol is adapted for our lab from the protocol included in the kit. You can find the full version of the protocol here. I recommend reading the section on the midi-prep before starting so you can learn what each of the components of the kit do.

<link to overnight wiki entry>

• Tube racks
• 50mL conical tube (Labeled: GREEN)
• 1.5mL micro-centrifuge tubes
• 50mL round bottom high speed conical tubes (purple lids, Labeled: BLACK, BLUE, RED)
• Pipette aide and serological pipettes
• High Speed Centrifuge (Eppendorf Centrifuge 5810 R, our lab)
  1. Place fixed angle rotor in the centrifuge with 50ml adapters
  2. Pre-chill centrifuge to 4C by using the “Fast Temp” option (setting temp to 4C) and close lid.
• Ice Bucket (with room for all of your tubes)
• Waste container (labeled WASTE 500ml beaker)

• Midi-prep Kit
  1. Buffer P1 is in the 4C refrigerator. If there is none you need to fix combine a vial of RNase A solution and Lyse Blue reagent to a new bottle of Buffer P1 and place that in the fridge.
• Isopropanol
• 70% Ethanol
• Baxter Sterile Water

1. Place 45mL overnight bacteria culture into labeled tubes (BLACK) and harvest by centrifuging samples at 6000xg for 15 minutes at 4°C
2. Remove supernatant
3. Resuspend the bacterial pellet in 4mL Buffer P1. When done with this step return to refrigerator.
4. Add 4mL Buffer P2 and mix by inverting samples 4-6 times; Do Not Vortex. The solution should be blue if Lyse Blue was added to Buffer P1.
5. Incubate at room temperature (15-25°C) for 5 minutes
6. Add 4mL pre-chilled Buffer P3 and mix by inverting samples 4-6 times or until the solution is colorless
7. Incubate on ice for 15 minutes
8. Centrifuge the samples at 18,000Xg for 1 hour at 4°C
9. Transfer the supernatant to clean, labeled tubes (BLUE)
10. Centrifuge the samples at 18,000Xg for 30 minutes at 4°C
11. During step 10, label and place a Qiagen Filter Tip with a blue holder over clean labeled tubes (GREEN)
12. Equilibrate by adding 4mL Buffer QBT to the filter and allow the column to empty by gravity flow
13. Apply the supernatant from Step 10 to the columns and allow it to flow through
14. Wash the column with 10mL Buffer QC twice, allowing it to completely flow through between washes
15. Place the columns over clean labeled tubes (RED)
16. Elute DNA with 5mL Buffer QF and allow it to flow through the column. When complete, remove the blue holder and column
17. Precipitate the DNA by adding 3.5mL ice cold Isopropanol and mix
18. Draw a line from the top of the tube cap down one side to the tip of the conical bottom. This should be facing out when you centrifuge
19. Centrifuge at 15,000xg for 1 hour at 4°C
20. Carefully decant the supernatant. Save off supernatant.
21. Wash the DNA pellet with 2mL 70% Ethanol by SLOWLY running the Ethanol down the OPPOSITE side of the tube as your line (and DNA)
22. Centrifuge at 15,000xg for 15 minutes at 4°C (make sure the line is facing out)
23. Carefully decant the supernatant. Save this supernatant.
24. Air-dry the pellet for 5-10 minutes (or until dry) and dissolve the DNA in 500µL dH2O
25. Spec sample on a nano-drop and record the information given, add them to the MM Mini-Preps and DNA spreadsheet and place in freezer in the appropriate box
    • If you have not been trained on the nano-drop ask for assistance.
26. Clean centrifuge, make sure to wipe down fixed angle rotor with 70% ethanol. Wipe all metal surfaces on the centrifuge itself as well with 70% ethanol, removing all salt from the machine. If you are unclear on what needs to be cleaned, please ask for directions.
27. Clean all areas you worked on, making sure to wipe down all surfaces with ethanol.