Team:iTesla-SoundBio/labnotebook/labnotebook061018.html

6/10/18

 

Purpose: To repeat the miniprep BBa_k608006 (biobricking backbone) for future digestion and ligation. The one conducted on 6/9/18 did not yield high enough concentrations.

 

Materials:

-inoculated BBa_k608006 [shaky]

-MX1 [Chilly, on the inside of the door]

-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]

-microcentrifuge tube

 

Procedure: (we have two kinds of cells to miniprep, 3 tubes of each kind of cell (BBa_k608006 and integration vector), so this procedure is being performed 6 times concurrently.)

 

1. Grow cells overnight ~5mL (done last night)
2. 
   Pellet cells in 1.5mL tube (centrifuge short)
   * Pour out supernatant

*Repeat step one until all liquid from culture tube has been transferred and centrifuged
3. Resuspend using 200uL MX1 buffer
4. Add 250uL MX2 -- DO NOT VORTEX -- gently invert tube 4-6 times
* Incubate at room temperature for 3 minutes
5. Add 350uL MX3 -- IMMEDIATELY -- gently mix as above
6. Centrifuge 10 minutes @ 15,000 rcf
* Place GenCatch plus column onto collection tube
7. Transfer the supernatant to the column using a P1000 micropipette set to 750 uL
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
12. Move column to new 1.5mL microcentrifuge tube.
*  Add 50uL Elution Buffer on CENTER of membrane
13. Stand column upright for 2 minutes @ room temperature
*  Centrifuge 30 seconds at 10,000 rcf

13a. Flick the tube, look at the flick of that wrist
14. Store @ -20C
15. Save the columns for reuse (new protocol for recharging the columns)

16. Put the miniprep vectors go back into the cylindrical box with the purple lid in Sniffles 1.

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 application from the desktop and enter the relevant information.