Team:iTesla-SoundBio/MinipreppingProtocolTemplate

 

Highlighted text are things that you need to fill in or change for your particular lab notebook depending on the variables, and concentrations.

Miniprepping [plasmid name(s)]

 

Materials:

• overnight cultures [plasmid name(s)]  [should be in shaky]
• MX1 [Chilly, on the inside of the door on the first shelf, keep cold, only take out when needed and return once done]
• MX2 [epoch kit under bench 4]
• MX3 [epoch kit under bench 4]
• Gencatch columns [epoch kit under bench 4]
• collection tubes [epoch kit under bench 4]
• Elution Buffer  [epoch kit under bench 4]
• sterile microcentrifuge tubes [on top of Shaky, inside autoclave-taped pipette boxes]

 

Procedure

1. Grow cells overnight ~3mL
1. Date/Time started: [date/time]
2. Plasmid Name 1, Qty X
2. Pellet cells in 1.5mL tube, pipetting 750 ul at a time:
1. Centrifuge short 1 min at 10,000 rcf
2. Pour out supernatant
3. Repeat step one until all liquid from culture tube has been transferred and centrifuged
3. Resuspend using 200uL MX1 buffer
1. IMPORTANT: Pipette up and down to resuspend, there should be no clump of cells after you are done
4. Add 250uL MX2 -- DO NOT VORTEX -- gently invert tube 4-6 times
1. Incubate at room temperature for 3 minutes
2. While you wait: Return the MX1 buffer to the fridge
5. Add 350uL MX3 -- IMMEDIATELY -- gently invert tube 4-6 times
6. Centrifuge 10 minutes @ 15,000 rcf
1. While you wait: Place GenCatch plus column onto collection tube
2. Reminder: be very gentle when you take the tubes out for the next step, as the white precipitate is contamination that is easily accidentally resuspended
7. Transfer the supernatant to the column using a P1000 micropipette set to 750 uL.
1. IMPORTANT: Be sure to avoid picking up any of the white precipitate, as that will contaminate your whole miniprep
8. Centrifuge 60 seconds @ 2,200 rcf -- DISCARD FLOW THROUGH
9. Add 500uL WN -> Centrifuge 60 seconds @ 7,000 rcf -- DISCARD FLOW THROUGH
10. Add 700uL WS -> Centrifuge 30 seconds @ 7,000 rcf -- DISCARD FLOW THROUGH
11. Centrifuge empty column 2 minutes @ 10,000 rcf
1. While you wait: Label the microcentrifuge tubes you’ll be using in the next step
12. Move column the new 1.5mL labelled microcentrifuge tube.
13. Add 30uL Elution Buffer on CENTER of membrane. Make sure not to puncture membrane.
14. Stand column upright for 2 minutes @ room temperature
1. While you wait: Move the collection tube to the “Used Miniprep columns” tube rack nearby the microwave
15. Centrifuge 30 seconds at 10,000 rcf
1. Move the used column to the “Used Miniprep columns” tube rack nearby the microwave
16. Flick the tube - as a prep step for reading the concentration on the NanoDrop
17. Store @ -20C in ____________ labelled as ______________.

Using the Nanodrop:

1. Plug NanoDrop USB into computer.
2. Open NanoDrop application (maybe?)
3. Use a p200 to put some amount of clean water on a wipe, then use said wipe to wipe off the black circle and the corresponding part on the lid. Keep the wipe around for reuse.
4. Pipette 0.65 uL of Elution Buffer into the middle of the black circle on the NanoDrop machine, then slowly close the arm.
5. Press “Blank” (in the application) to blank the NanoDrop (this gives it a baseline from which to measure your sample’s concentration).
6. After it’s finished blanking, wipe the NanoDrop off with the wipe from earlier.
7. Pipette 0.65 uL of [whatever is being measured] into the NanoDrop as before. If you’ve just done the miniprep or taken the tubes out of the freezer, make sure to flick them a few times to a more consistent concentration. Close the lid.
8. Label the sample in the “Sample ID” box.
9. Press “Measure.”
10. Get the result. The graph should (hopefully) look something like this:
11. Take a screenshot and save it, naming it like so: [sample name] [today’s date]
12. Open the NanoDrop Results google form application from the desktop and enter the relevant information and upload the screenshot.

 

Results

Label	Concentration	Volume	Location	Notes