Biomod/2013/Harvard/protocols

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Protocols

Agilent Herculase II PCR Amplification

Protocol:

  1. In a PCR tube, mix (50 µL final volume):
    1. 35 µL water
    2. 10 µL HercII Buffer (5X)
    3. 0.5 µL dNTP (25mM each, 100mM total final)
    4. 1.25 µL forward primers (final conc?)
    5. 1.25 µL reverse primers (final conc?)
    6. 1 µL Template DNA (diluted to 10 ng/µL)
    7. 1 µL HercII DNA polymerase
  2. Give the tubes a brief spin
  3. Thermocycling conditions:
    1. Step 1: 95˚C, 2:00
    2. Step 2 X30
      1. Phase 1: 95˚C, 0:15
      2. Phase 2: (Tm - 5)˚C, 0:20
      3. Phase 3: 72˚C, 0:30 per 1kb
    3. Step 3: 72˚C, 3:00

Bacterial Cell Cloning

Entire procedure is done under flame

  1. For small test tubes add:
    1. 5mL LB media
    2. 50µL 20% glucose (final conc. 0.2%)
    3. 7.35µL of CM (final conc. 50µg/mL, working stock: 34 mg/mL)
  2. Add the cells
    1. If from glycerol stock, stab with pipette tip.
    2. If from LB-agar plate, pick the colony (mark it on the cap) and stab the colony
  3. Incubate on shaker overnight


ZYMO DNA Clean & Concentrator

ZYMO DNA Clean & Concentrator - 5 Kit was used to purify DNA from PCR.

Protocol:

  1. Add 2-7 Volumes of DNA Binding Buffer to each volume of DNA sample.
    1. For plasmid or genomic DNA 2kb, add 2 volumes.
    2. For PCR or short DNA fragments, add 5 volumes.
    3. For ssDNA purification, add 7 volumes
  2. Load the mixture into a Zymo-Spin Column in a Collection Tube.
  3. Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard flow through.
  4. Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds.
  5. Repeat previous step, this time centrifuging for 1 minute.
  6. Place the Zymo-Spin Column into a new 1.5 ml tube. Add ≥6 µl of DNA Elution Buffer or water directly to the column matrix and spin (30 seconds) to elute the DNA.

Bacterial Cell Culture Solutions

LB media

  1. For 1L of LB media add:
    1. 10g Tryptone
    2. 10g NaCl
    3. 5g Yeast extract
    4. Fill to 1L with DI water
  2. Autoclave at P6 setting

LB-Agar Plate media

  1. For 1L of LB-Agar media, mix:
    1. 960mL water (860 mL if adding glucose)
    2. 10g Tryptone
    3. 10g NaCl
    4. 5g Yeast extract
    5. 15g Agar (add agar last)
  2. Autoclave (liquid option): 30 min. sterilization / 20 min drying
  3. Cool to 50˚C in water bath
  4. Next three steps are under flame
  5. (if adding glucose: add 100mL of 20% glucose, final conc. 2% glucose)
  6. Add antibiotics to the specified final conc:
    1. chloramphenicol: 50 µg/mL
    2. ampicillin: 100 µg/mL
    3. Tetracycline: 50 µg/mL
  7. Pour onto the petri dishes (pre-marked by type: two-black lines=CM, red-line=glucose)


BLACaM Assays

Chemically Competent Cells Transformation

This protocol uses NEB Chemically Competent Cells (ie Turbo Competent E. coli High Efficiency)

Protocol:

  1. Thaw chemically competent cells on ice.
  2. Transfer 50 µl of competent cells to a 1.5 ml microcentrifuge tube (if necessary).
  3. Add 2 µl of assembled product to NEB competent cells.
  4. Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.
  5. Heat shock at 42°C for 30 seconds. Do not mix.
  6. Transfer tubes on ice for 2 minutes.
  7. Add 950 µl of room temperature SOC media to tubes.
  8. Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  9. Warm selection plates to 37°C.
  10. Spread 100 µl of the cells onto the plates with appropriate antibiotics. Use amp plates for positive control sample.
  11. Incubate plates overnight at 37°C.

Qiagen MiniPrep

For overnight cell cultures 1~5mL of E. Coli in LB medium:

  1. Transfer cell culture into centrifuge tubes
  2. Centrifuge for 5 min. at 4,000 rpm
  3. Discard the supernatant
  4. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
  5. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Do not vortex. Continue inverting the tube until the solution becomes viscous + slightly clear blue. Do not allow lysis reaction to proceed for more than 5 min.
  6. Add 350 µl Buffer N3 and mix immediately + thoroughly by 4-6x inverting the tube. The solution should become cloudy.
  7. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  8. Apply the supernatants from the previous step to the QIAprep spin column by decanting or pipetting.
  9. Centrifuge for 60 s. Discard the flow-through.
  10. Wash QIAprep spin column by adding 750µL Buffer PE and centrifuging for 60 s.
  11. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
  12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

NanoDrop

  1. Wash the
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