Lab Notebook

There was insufficient time to order parts or run wet lab experiments but if this project were to be continued then these would be the procedures most likely used.

NEB In vitro digestion of DNA with Cas9

Nuclease, S. pyogenes (M0386)

Purpose: To check for cas9 cutting and binding to target DNA in an in vitro enviroment.

Protocol:

1. Assemble the reaction at room temperature in the following order:

   Nuclease-free water

   NEBuffer r3.1

   300 nM sgRNA 3

   1 µM Cas9 Nuclease, S. pyogenes (M0386S)

   Reaction volume

   20 µl

   3 µl

   µl (30 nM final)

   1 µl (~30 nM final)

   27 µl

   Pre-incubate for 10 minutes at 25⁰C

   30 nM substrate DNA

   Total reaction volume

   3 µl (3 nM final)

   30 µl

2. Mix thoroughly and pulse-spin in a microfuge.

3. Incubate at 37°C for 15 minutes.

4. Add 1 μl of Proteinase K to each sample, Mix thoroughly and pulse-spin in a microfuge.

5. Incubate at room temperature for 10 minutes.

6. Proceed with fragment analysis.

Notes: N/A

Next: Plasmid miniprep to start in vivo tests.

Plasmid Miniprep

Purpose: To extract plasmid DNA for in vivo tests

Protocol:

1. Gently swirl the contents of the culture tube to resuspend the cells. Transfer to a 15 mL centrifuge tube and spin for 5 min to pellet the cells.
2. Label a 1.5 mL tube. Add 200uL solution 1, resuspend the cells, and pipet the cell suspension into the microfuge tube.
3. Close the caps and place the tubes in a centrifuge (remember to balance the centrifuge by putting two tubes opposite one another) and spin at maximum speed for 20 s. After the spin, you should see amber liquid and a white pellet.
4. Withdraw and discard the supernatant using a pipettor, being careful not to disturb the cell pellet. Discard the supernatant in a waste container.
5. Add 200 uL of Solution 1 to each tube and resuspend the cells by vortexing. It's very important that the cell suspension is homogenous and no clumps are visible. The cells are now resuspended in a buffered solution with RNase.
6. Add 200 uL of Solution 2 to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. DO NOT VORTEX.
7. Add 200 uL of ice-cold Solution 3 to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. A white precipitate will form.
8. Place the tubes in a centrifuge (balanced) and spin at maximum speed for 5 minutes. The precipitate will pellet along the side of the tube.
9. Transfer the supernatants into clean 1.5 mL tubes, being careful not to pick up any of the precipitate. Discard the tubes with the precipitate and KEEP the tubes with the supernatant.
10. To each tube of supernatant add an equal volume (about 600 uL) of isopropanol. Close the caps and mix vigorously. Let the tubes stand at room temperature for 2 minutes, place them, with their hinges pointing outward from the center, in a centrifuge (balanced) and spin at maximum speed for 5 minutes.
11. Carefully remove and discard the supernatant.
12. Add 200 uL of ice cold absolute ethanol to each tube and mix by inversion several times.
13. Spin the tubes at maximum speed in a centrifuge for 2-3 minutes (hinges out).
14. Carefully remove and discard the supernatant. Try to get as much out as possible without dislodging the pellet of plasmid DNA.
15. Leave tubes open to let ethanol evaporate. When the ethanol is gone add 20 uL of nuclease free water to dissolve the pellet. Pipet the 20 uL in and out, up the side of the tube to ensure that all of the plasmid DNA comes into contact with the TE buffer.
16. Store your miniprep DNA in the freezer.

Notes: N/A

Next: Insert custom sequence into the plasmids and insert plasmids into lambda phages.

Later experiments could also include testing transformed phages on resistant E. coli to test for success.

Protocols from:

1. https://www.neb.com/en-us/protocols/2014/05/01/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyogenes-m0386
2. Modified from http://csmbio.csm.jmu.edu/biology/courses/bio480_580/mblab/miniprep.html