Team:Washington/Project/Protocols/Kunkels

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Contents

Kunkel's Mutagenesis

Produce ssDNA

1. Produce ssDNA (RECOMNEDED TO DO IN DUPLICATE! SEE NOTE AT END OF SECTION 2)

  • a. Transform plasmid into chemically competent CJ236 cells.
    • i. -Plate onto a Chlor+YOUR ANTIBIOTIC plate and incubate overnight at 37C.
  • b. Inoculate 6 colonies into 3ml of LB + YOUR antibiotic. (NO chlor here per NEB instructions)
  • c. Grow for 4-6 hours at 37C, shaking at 200rpm (until cloudy).
  • d. Add 3ul of M13K07 helper phage.
  • e. Continue growing for 1 hour at 37C, shaking at 200rpm.
  • f. Expand culture by diluting 1ml into 50ml of LB + antibiotic in 250ml flask.
  • g. Grow overnight at 37C, shaking at 200rpm.

Harvesting ssDNA

2. Harvesting ssDNA

  • a. Spin down overnight culture in sterile 50ml Falcon tube at 7000rpm for 20 minutes at 4C.
  • b. Transfer supernatant (contains phage) to a new sterile 50ml Falcon tube.
  • c. Add 10ml 20% PEG/2.5M NaCl and mix thoroughly and incubate on ice for 45 minutes.
  • d. Spin down the phage at 7000rpm for 20 minutes at 4C.
  • e. Decant liquid and let tube stand upright to drain off the rest of the liquid.
  • f. Resuspend the pellet in 2ml 1xPBS. Vortexing is okay.
  • g. Transfer the 2ml into two microfuge tubes (will have 1ml in each).
  • h. Spin at 14,000rpm for 5 minutes.
  • i. Transfer the supernatant to two new microfuge tubes, each with 300ul PEG/NaCl.
  • j. Vortex and incubate at room temperature for 10 minutes.
  • k. Spin down phage at 14,000rpm for 2 minutes.
  • l. Pipette off supernatant. Do a second quick spin to collect residual liquid and pipette it off.
  • m. Resuspend the pellet (phage) in 1ml 1xPBS. Use that 1ml to resuspend the other pellet.
  • n. Spin down at 14,000rpm for 5 minutes.
  • o. Transfer the supernatant (phage) to a new microfuge tube.
  • p. Harvesting ssDNA from Phage using Qiagen Qiaprep M13 kit (#27704)
    • i. DO NOT SPIN OVER 8000rpm when using this kit!
    • ii. Final solution should be greater than 20ng/uL. If not try again; ssDNA can randomly fail so often it is best to make 2 batches in parallel (works as a nice counter balance as well).

Kinase oligo

3. Kinase oligo in a 96 well plate

  • a. Design mutagenic oligo using statagene’s primer design and order the 5’->3’ antisense oligo
  • b. Make Kinase Reaction Mix (make #rxn+2)
    • i. 3uL Kinase Buffer/rxn
    • ii. 1uL of 10mM ATP/rxn
    • iii. 1uL T4 Polynucleotide Kinase/rxn
    • iv. 18uL ddiH2O/rxn
  • c. Combine oligo and reaction mix in 96well PCR Plate
    • i. Aliquot 23uL of Kinase Reaction Mix using repeater
    • ii. Spin down (Plates use 4000rpm for 2min in swinging bucket, or Strip tubes use BenchTop for 10sec)
    • iii. Add 7uL of 100uM oligo to the BOTTOM of each well and pipette up and down
  • d. Cover AlumiSeal (plates) or caps (strip tubes)
  • e. Spin down (Plates use 4000rpm for 2min in swinging bucket, or Strip tubes use BenchTop for 10sec)
  • f. Gently mix (Plate mixer for 30sec @ 1650)
  • g. Incubate at 37deg C in PCR machine for 1 hour
  • h. Spin down (Plates use 4000rpm for 2min in swinging bucket, or Strip tubes use BenchTop for 10sec)
    • i. Store on ice short term, or -20 long term
    • ii. These can be re-used in the future so no need to throw them away!

4. Dilute Mutagenic Kinased Oligo (Optimal molar ratio is 1:4 dU-ssDNA:Oligo)

  • a. Determine molarity of dU-ssDNA
    • i. Xng/uL * 105nM nucleotides/33ng/uL * 1/#bp’s = nM ssDNA

5. 100ng/uL of pET29b+ with 1kb insert (~6kb) is about 50nM of dU-ssDNA

  • b. In a fresh PCR plate dilute Kinased Oligo so 10x the concentration of dU-ssDNA
    • i. Transfer XuL of oligo to a fresh plate with 200uL of ddiH20 aliquot into each well. After transfer mix with P200 (set at 100uL) to ensure primer is homogenous.
    • ii. EXAMPLE CALCULATION:

6. Determine Oligo Dilution: 100uM oligo * 7/30 * x/100 = 10 * (nM dU-SSDNA) * 1uM/1000nM

  • a. For above approximation of 100ng/uL of dU-ssDNA: x ~= 2uL
    • iii. If doing multiple mutations (>1 oligo) add xuL of EACH oligo to this dilution!

Anneal Diluted Oligo and ssDNA

5. Anneal Diluted Oligo and ssDNA

  • a. ORDER IS IMPORTANT HERE FOR PROPER MIXING!
  • b. Combine 0.2uL of T4 DNA Ligase Buffer with 2uL of ssDNA, (make #rxn+2)
  • c. Aliqout 2.2uL of the mix into a in a fresh PCR plate or strip tubes
  • d. Spin down (Plates use 4000rpm for 2min in swinging bucket, or Strip tubes use BenchTop for 10sec), also SPIN DOWN FROZEN PRIMERS IF IN PLATE
  • e. Add 2uL of THE DILUTED kinased primer (single or mixed) to generate desired mutant
    • i. Always do a background as well where you have ssDNA with no oligo. This will allow to you to estimate your mutation efficiency and know the approximate number of colonies you need to screen in order to find the mutation desired.
    • ii. Add to the BOTTOM (not side) of the plate and PIPPETE UP AND DOWN
  • f. Seal with AlumiSeal (plate) or Caps (Strip Tubes)
  • g. Gently vortex or mix in Plate mixer (30sec @ 1650)
  • h. Run Jasmine→905020program in PCR machine (USE heated lid)
  • i. 95 2min, 50 2min, 20 forever

Polymerize DNA

6. Polymerize DNA

  • a. Make polymerization reaction mixture (make #rxn+2)
    • i. 0.6uL 10x T4 Ligase Buffer/rxn
    • ii. 0.4uL 25mM dNTPs/rxn
    • iii. 0.4uL 10mM ATP/rxn
    • iv. 0.4uL T7 Polymerase (unmodified from NEB)/rxn
    • v. 0.4uL T4 Ligase/rxn
  • b. Add 2.2uL of polymerization reaction mixture to each annealing reaction
  • c. Seal with AlumiSeal (plate) or Caps (Strip Tubes)
  • d. Spin down (Plates use 4000rpm for 2min in swinging bucket, or Strip tubes use BenchTop for 10sec)
  • e. Gently mix (plate mixer for 30sec @ 1650)
  • f. Incubate at room temperature for 1+ hrs.

Transform

7. Transform

  • a. Associate DNA to Cells
    • i. Place reactions on ice for 5min
    • ii. Add 50uL of standard CCMB prepped BL21(DE3)* to each well
  • b. Only take the cells out of the -80 approx 10min before adding to reaction. KEEP ON ICE AT ALL TIMES. Other cells can be used as well, BL21(DE3)* are only needed if protein of interest is under a T7 promoter.
  • c. Seal with AlumiSeal (plates) or caps (strip tubes)
  • d. Gently mix (plate mixer for 30sec @ 1650)
    • i. Make sure there are no bubbles, may have to use pippettor to remove bubbles.
    • ii. Do not remove from ice for more than a minute
  • g. Keep Cells on ice for 5min
  • h. Preheat PCR Block to 42Deg (incubate mode)
  • I. HeatShock
    • i. Quickly transfer plate or strips to a preheated PCR Block (42deg)
    • ii. Let sit at 42deg for 60sec
    • iii. Quickly transfer back to ice bucket
    • iv. Let equilibrate for 2min
  • j. Recover
    • i. Aliquot 200uL of LB/Glucose into each tube
  • k. If tubes are sealed with alumiseal spin down before removing, otherwise cross contamination will occur!
  • l. LB/Glucose =1mL LB with 10uL of 2M Glucose will work. But any rich media should be fine
    • ii. Seal with alumiseal or caps
    • iii. Incubate for 1hr at 37deg
  • m. Plate Reactions
  • n. On individual plates (not sure how to get around this) plate out ~200uL of each reaction
  • o. I find it works best to do 12 plates at a time
  • p. Add ~10 glass beads/plate
  • q. Add the 200uL of solution
  • r. Shake to spread
  • s. Turn plate and tap so beads are transferred to the lid, than pour out beads into 70% EtOH filled beaker.
  • t. Move to the next row.
    • ii. Dry plates at 37deg for ½ hr, lid-side up.
    • iii. Turn plates so agar side is up and incubate at 37deg overnight


Solutions

20%PEG/2.5M NaCl

  • 200g PEG (polyethylene glycol) 8000MW
  • 141.6g NaCl
  • Add dH20 to 1L
  • Autoclave with a stir bar, stir immediately after autoclaving until cool, and store at 4C.

1xPhosphate Buffered Saline (PBS)

  • 800ml dH20
  • 8g NaCl
  • 0.2g KCl
  • 1.44g Na2HPO4
  • 0.24g KH2PO4
  • Adjust pH to 7.4
  • Add dH2O to 1L
  • Autoclave



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