EspP Autotransporter

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Protocol for making EspP Autotransporter


Contents

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.

Cloning by PCR

This is the basic PCR method to amplify your DNA from a plasmid or genomic DNA sample using the Expand polymerase. You also use this protocol for an EIPCR reaction.

The oligo concentrations in your stocks should be 100uM. You use them at 10uM in this protocol. So, you 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:

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

The Expand temperature programs are a little complicated, so I won’t write them out here. They all involve 2 distinct cycling phases—the first 10 cycles have shorter extensions than the latter 20 cycles. You choose which one you want based on the length of your predicted product. If it is under 2kb, use 2K55. If it is between 2kb and 4kb, use 4K55. If it is over 4kb, use 8K55. In each case, start with the "55" version of the program. If you get no product or poor yield of product, repeat the same PCR reaction as two different variants, both with the “45” program. The first reaction will be the same composition as the first. For the second one, don’t add 3.3 uL of the water and instead use the volume for 3.3 uL of DMSO (dimethylsulfoxide). If these PCRs fail as well, well you'd either redesign, use a different template source, or give up.

Regular Zymo Cleanup (1)

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.
  2. Transfer into the Zymo column (small clear guys)
  3. spin through, 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

EcoRI/BamHI Digest of PCR Products

For PCR products, you will only digest a portion of your purified PCR product. Note that you must make a minor modification to the procedure if your DNA is shorter than 300bp (you add a little isopropanol to the ADB after melting).

  • 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
  • If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column

Regular Zymo Cleanup (2)

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.
  2. Transfer into the Zymo column (small clear guys)
  3. spin through, 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

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.

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?

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