Team:TP-CC San Diego/Protocols

Protocols

Protocols

TRANSFORMATION PROTOCOL

Procedure:

  1. Thaw comp cells on ice for 10 mins.
  2. Add 10uL of DNA to comp cells.
  3. Let sit on ice for 10 mins
  4. Heat shock at 42˚C for 1 min
  5. Let sit on ice for 2 min
  6. Add 300uL of SOC media
  7. Put in 37˚C shaker at 200 rpm for 1 hr
  8. Place agar plates in 37˚C incubator to warm up; remove when needed.
  9. Pipette 300uL of comp cells onto agar plate
  10. Use sterilized beads to spread comp cells evenly
  11. Place in 37˚C incubator overnight

INOCULATION PROTOCOL

Procedure:

  1. Add 5mL of LB to Polypropylene round bottom tube
  2. Add 5uL of Carb antibiotic to LB
  3. Label and pick colony from plate
  4. Loosely secure cap to allow air flow
  5. Place in 37˚C shaker at 200rpm overnight

GUIDE RNA DESIGN

Procedure:

  1. Find gene of interest
  2. Used exon 1 and exon 8 of EGFR gene because exon 2-7 has mutations
  3. Input into crispr gRNA design tool: http://crispr.mit.edu
  4. Review possible off target sights and mismatches
  5. Choose guides that have less off target sights and all sights have at least 3 mismatches

DIGESTION PROTOCOL

Materials:

  1. 5ug TLCV2
  2. 3uL FastDigest BsmBi
  3. 3uL FastAP
  4. 6uL 10X FatDigest Buffer
  5. 0.6uL 100mM DTT(freshly prepared)
  6. 32.4uL ddH2O

Procedure:

  1. Combine all materials in a test tube.
  2. Digestion 2. Place in 37˚C water bath for 1 hour

LIGATION PROTOCOL

Materials:

  1. 1uL cut plasmid
  2. 1uL oligo
  3. 1uL 10X T4 ligation buffer
  4. 6uL H2O
  5. 1uL Ligase

Procedure:

  1. Combine all materials in a test tube.
  2. Place at room temperature for one hour.

MINIPREP PROTOCOL

Procedure:

  1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting.
  7. Centrifuge for 30–60 s and discard the flow-through.
  8. Wash the QIAprep 2.0 spin column by adding 0.75ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep 2.0 spin column to the collection tube. Centrifuge for 1 min to remove residual wash buffer.
  9. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

ANNEALING PROTOCOL

Procedure:

  1. Place reaction in a thermocycler under following conditions:
  2. 37˚C 30 mins
  3. 95˚C 5 mins and then ramp down to 25˚C at 5˚C/min
  4. Before Ligation, dilute annealed oligos at 1:200 in EB

qPCR PROTOCOL

Procedure:

  1. H2O 4uL
  2. 2x PCR Mixture 10uL
  3. Primer Mixture 4uL
  4. Template DNA 2uL
  5. Set thermocycler to following conditions:
    • 95˚C 5min
    • 95˚C 15sec
    • 60˚C 30sec
    • 72˚C 15sec
  6. Repeat steps 2-4 for 30 Cycles
  7. 12˚C hold