Myc Tag

LR Gateway Transfers

There are no restriction enzymes are ligases in the Gateway reaction! You just mix plasmids and enzyme, let it cook, then transform

• Set up the following mixture in a 0.5mL microcentrifuge tube

 3uL ddH2O
 0.5uL Donor plasmid (a pBca1256-*)
 0.5uL Recipient plasmid (a pBca1254**)

• Add 1uL of LR Clonase
• Slam on bench upside down to mix
• Quick spin to send it to the bottom of the tube
• Incubate at 25 degrees on the thermocycler for 1hr
• Add 0.5uL proteinase K
• Slam on bench upside down to mix
• Quick spin to send it to the bottom of the tube
• Incubate at 37 degrees on the thermocycler for 10min.
• Put on ice, proceed to transformation

Transformation by heat-shock

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock.

  1. Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water
  3. Add 30 uL of KCM salts
  4. Put your ligation mixture on ice, let it cool a minute or two
  5. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 2 min at 42
  8. Put back on ice for 1 min
  9. For ampicillin selection, you can plate immediately, otherwise:
 10. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
 11. Plate on selective antibiotics, let incubate overnight

Picking of colonies

   * For each construct you will pick and later miniprep 2 colonies
   * Add 4mL of LB media with the appropriate antibiotics to a clean test tube
   * Pick a well-isolated, round, and "normal" looking colony with a toothpick
   * Drop it in the test tube
   * Incubate at 37 overnight 

Miniprep purification of DNA

MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)

  1. Pellet around 1.5 mL or 2 mL saturated culture by spinning full speed, 30 seconds.
  2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
  4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
  5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
  6. Spin in centrifuge at top speed for 5 minutes.
  7. Label blue columns with an alcohol-resistant lab pen.
  8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
  9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
 10. Wash each column with 500 uL of PB buffer.
 11. Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
 12. Wash with 750uL of PE buffer (washes the salts off the resins).
 13. Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
 14. Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
 15. Label new tubes and put columns in them.
 16. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
 17. Spin in centrifuge at top speed for 30 seconds.
 18. Take out columns and cap the tubes.
 19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

RESTRICTION DIGEST CHECK

Set up the following 10uL reaction in a PCR tube:

6.5 uL ddH2O
2uL Miniprepped plasmid
1uL 10x NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)

• Incubate at 37 on the thermocycler for 30 minutes
• Run an analytical gel
• Take a picture of the gel
• Calculate the expected fragment sizes
• Are the calculated sizes consistent with the bands on the gel?