Protein A FLAG Tag M10018 M10019

Protocol for making Protein A FLAG Tag M10018 M10019

GOOD LUCK!

Oligos

- The initial oligos will be at 28.8 nanoM. You want a conc. of 100nM give or take. - Add 288ul of water. This makes the conc. 100nM of the oligos
Protip
- Spin down oligos before adding water to make sure it is at the bottom. due the crappy spinner, no balancing required.
- Mix (by scrapping it against the tube rack ---> high tech!) then spin down again.

Wobble

• For our 100nm stock:

29 uL water
5 uL Expand 10x Buffer 2
5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
5 uL Oligo 1 (100uM)
5 uL Oligo 2 (100uM)
0.75 uL Expand Polymerase 1

• The wobble program should be preset. Just in case, here's the settings:

2 min at 94
10 cycles of:
30 sec at 55
30 sec at 72
(or something similar)

Protip
No gel is required because your pieces are too small to see.
Your next step is "special" small frag cleanup as oppose to the large frag. This removes the dna polymerase.

Small-Frag Zymo Cleanup

• The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest reaction:

  1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
  2. Transfer into the Zymo column (small clear guys)
  3. Add 500uL of Ethanol and pipette up and down to mix
  4. spin through, discard waste.
  5. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
  6. spin through, discard waste.
  7. Add 200 uL of PE or Zymo Wash buffer
  8. spin through, discard waste.
  9. spin for 90 seconds, full speed to dry.
 10. elute with water into a fresh Eppendorf tube

EcoRI/BamHI Digest of Wobble Products

For wobble products, you will digest the entire extension reaction-worth of DNA Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.

• Set up the following reaction in a PCR tube:

 8uL of eluted PCR product
 1uL of NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI

• Incubate at 37 degrees on the thermocycler for 1hr

Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
Incubate the reaction at 37 degrees on the thermocycler
Proceed to another Zymo small fragment cleanup
Protip
The official directions calls for 50 ul which is a different conc. than we want for our reaction.

Ligation of EcoRI/BamHI digests

• Set up the following reaction:

6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase

• Pound upside down on the bench to mix
• Give it a quick spin to send it back to the bottom of the tube
• Incubate on the benchtop for 30min
• Put on ice and proceed to the 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.