Team:iTesla-SoundBio/labnotebook/labnotebook060918.html

6/9/18

 

Purpose: To miniprep integration vector and BBa_k608006 (biobricking backbone) for future digestion and ligation. Also, to run an analytical gel on last night’s PCR to analyze its success.

 

Materials:

-inoculated BBa_k608006 and integration vector [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
   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
   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
   14. Store @ -20C
   15. Save the columns for reuse (new protocol for recharging the columns)
3. 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.

 

Analytical Gel:

 

Materials:

-TAE Solution [carboy near sink]

-250mL flask [SoundBio storage]

-Loading Dye [gel cart by bench 1]

- 20,000X Gel stain [foil envelope in Chilly]

-Parafilm [next to microwave]

-scale and weigh boat

-Graduated cylinder [storage]

-gel comb, gel tray (10 wells) [gel cart by bench 1]

-power supply [gel cart]

 

Procedure:

1. Measure out 30mL TAE in a graduated cylinder.
2. Measure out 0.3 g of agarose using a weigh boat and the the scale
3. Pour 15mL from graduated cylinder into the flask.
4. Add the 0.3 g of agarose into the flask, and add rest of the TAE.
5. Microwave flask for 30 secs, and then microwave in 10 sec intervals until there is no more agarose visible, and it hasn’t started bubbling. Make sure to place some paper towel in the open end of the flask so it doesn’t bubble over.
6. Allow flask to cool, swirling with a glove every couple minutes to prevent solidification.
7. Pipette 1.5 uL of 20,000X gel stain into the cooled flask.
8. Pour contents of flask into gel tray until it reaches the tip of the gel tray to avoid flowover and wait for it to solidify (~30 minutes). Tape the ends of the gel tray to prevent the solution from flowing out. Remove tape and gel comb after solidification.
9. Place gel tray in gel box and fill gel box with TAE solution until gel tray is submerged.
10. Pipette the following reagents (in table) onto parafilm in the order they will appear on the gel. Pipette up and down to mix. Take care to remember which tube corresponds to which droplet.
11. Load from parafilm into wells using the following diagram:

<tbody> </tbody>

1	2	3	4	5	6	7	8
	3uL pcr purified 2 +2uL LD		3uL pcr purified C + 2uL LD		3uL pcr purified 1 + 2uL LD		3uL 10kb ladder + 3uL LD

[This order is a little odd, which is for two reasons: firstly, we accidentally put C where 2 was originally supposed to be, and secondly, we flipped the gel in the gel tray.

 

12. Connect to power supply and turn it on at 140 V
13. Wait until orange band is ? of the way down the gel and then shut off
14. Once gel has finished running, view it under the blue light reader.