Hydroxyapatite-binding peptide

Entire protocol for constructing the Hydroxyapatite-binding peptide (M10027) using EcoRI and BamHI This part will go into pBca9495AK-Bca1144#5 and will be synthetically created using the wobble reaction This is a passenger protein of the type { }

OLIGOS
- they will be at a concentration of 28.80 nmol (may be at a diff concentration, need to check) and we want a final concentration of 100uM --> add 288 ul of water (if 28.80 nmol) (make sure you spin down the tubes before adding the water to make sure everything's at the bottom); mix well (tube rack) and spin down again --> oligo concentrations are now 100uM

WOBBLE REACTION
29 uL water 5 uL Expand 10x Buffer 2 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) 5 uL Ost003F (100uM) 5 uL Ost004R (100uM) 0.75 uL Expand Polymerase 1
 * Prepare the following reaction:

2 min at 94 10 cycles of: 30 sec at 55 30 sec at 72 (or something similar)
 * Run the wobble program, which is:
 * There is no point in running an analytical gel afterwards, there is nothing to see
 * You'll want to run short fragment cleanups to remove the polymerase prior to digestion steps

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 (50ul) into a fresh Eppendorf tube

DIGEST
50uL of eluted PCR product 5.7uL of NEB Buffer 2 1uL EcoRI 1uL BamHI
 * Set up the following reaction:
 * Incubate at 37 degrees on the thermocycler for 1hr

ANOTHER SMALL FRAG ZYMO CLEAN-UP
- elute in 50ul

LIGATION
6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL pBca9495AK-Bca1144#5 1uL Insert digest 0.5uL T4 DNA Ligase
 * Set up the following reaction:
 * 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
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 37C overnight

MINIPREP PURIFICATION OF DNA
1. Pellet 1.5 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 MAPPING
3uL of miniprep DNA 5ul H20 1uL of NEB Buffer 2 0.5uL EcoRI 0.5uL XhoI lanes 1-2: Hydroxyapatite BP lanes 3-4: Magnetic Bead BP for upaG: --> send in for sequencing
 * Incubate at 37 degrees on the thermocycler for 1hr
 * run out on a gel to check size (should be 2026bp, 1196bp)