UpaG Autotransporter short version

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Entire protocol for constructing the upaG Autotransporter short version basic part (M10026) using EcoRI and BamHI
This part will go into pBca9495KC-Bca1144#5 and will be PCR-ed off of CFT073 genomic DNA
This is an N-terminal display protein of the type {<part!}
Since this piece is 298bp (on the 300bp borderline), the following protocol treats it as a regular, large DNA piece, not a small fragment.

Contents

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

PCR

- first need to make an oligo dilution of:
   9uL Water
   1uL 100uM oligo
You can throw away the remainder of the diluted oligo when you are done, but hold onto your stock tube!

Set up the following reaction in a PCR tube (this is only for regular PCR, not Wobble Reaction):

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Ost001F, 10uM
1uL Ost002R, 10uM
0.5uL Expand polymerase "1"
0.5uL CFT073 genomic DNA

Use C55 (since it's 298 bp)

GEL

- 5ul sample + 1ul dye
- ladder: 3-5ul
- run at 100V for about 20 minutes
- gel looked good

Image:upaG.jpg
upaG 300bp

REGULAR ZYMO CLEANUP

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction.  It also will
remove the buffer and restriction enzymes from a restriction digest reaction.

1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction (min. is 3 volumes)
2. Transfer into the Zymo column (small clear guys)
3. spin through (15 secs at max speed, ie around 15,000 rpm), discard waste.
4. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
5. spin through, discard waste.
6. Add 200 uL of PE or Zymo Wash buffer
7. spin through, discard waste.
8. spin for 90 seconds, full speed to dry.
9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction (used 33ul)

DIGEST

*Set up the following reaction:
  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

REGULAR ZYMO CLEANUP

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction.  It also will
remove the buffer and restriction enzymes from a restriction digest reaction.

1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction (min. is 3 volumes)
2. Transfer into the Zymo column (small clear guys)
3. spin through (15 secs at max speed, ie around 15,000 rpm), discard waste.
4. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
5. spin through, discard waste.
6. Add 200 uL of PE or Zymo Wash buffer
7. spin through, discard waste.
8. spin for 90 seconds, full speed to dry.
9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction (used 10ul)

LIGATION

*Set up the following reaction:
  6.5uL ddH2O
  1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
  1uL pBca9495KC-Bca1144#5
  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

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 BamHI
*Incubate at 37 degrees on the thermocycler for 1hr
*run out on a gel to check size (should be 2974bp, 282bp)

--> send in for sequencing

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