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