Team:TP-CC San Diego/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

Procedure:

  1. 5ug TLCV2
  2. 3uL FastDigest BsmBi
  4. 3uL FastAP
  5. 6uL 10X FatDigest Buffer
  6. 0.6uL 100mM DTT(freshly prepared)
  7. 32.4uL ddH2O

ANNEALING PROTOCOL

Procedure:

  1. Add 5mL of LB to Polypropylene round bottom tube

LIGATION PROTOCOL

Procedure:

  1. Add 5mL of LB to Polypropylene round bottom tube

SPIN PROTOCOL

Procedure:

  1. Add 5mL of LB to Polypropylene round bottom tube