User:James Chappell / Protocols

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Transformation Protocol 1

The aim of this protocol is to access whether we can successfully transform B.subtilis with the integration vector pDR110.

Reagents

1. Minimal salts (x5)(per litre):

  • (NH4)2SO4 10g
  • K2HPO4 74g
  • KH2PO4 27g
  • Trisodium citrate 9.5g
  • MgSO4.7H2O 1.0g pH7.0.

2. Minimal-growth medium (per 100ml)(20ml needed for this protocol):

  • 20ml minimal salts
  • 2.5ml glucose
  • 1ml casamino acids
  • If required, supplement with appropriate growth factors (amino acids and nucleotides, 20µg/ml; vitamins, 0.5µg/ml).

3. Starvation medium (per 100ml) (11ml needed for this protocol):

  • 20ml minimal salts
  • 2.5ml glucose

4. LB media (per 1 Litre)(10ml needed for this protocol)(:

  • 10g Bacto-tryptone
  • 5g yeast extract
  • 10g NaCl pH 7.5

5. LB agar plates

  • 1 plate containing of Spectinomycin 100ug/ml

Protocol

Day 1

  1. Pick a single colony from the B.subtilis plate and inoculate 10ml of minimal growth medium in a 100ml flask. Grow overnight at 37oC in a shaking incubator at ~200rev/min.

Day 2

  1. Add 1.4ml of overnight culture to 10ml of pre-warmed fresh minimal-growth medium (plus growth factors)in a 100ml flask.
  2. Grow for 3 hours at 37ºC in shaking incubator at ~200rev/min.
  3. Add 11ml of starvation medium and continue growth for 2 hours at 37ºC with increased agitation at ~ 300rev/min.
  4. The culture is now maximally competent for about half an hour,
  5. Sterile glycerol is added to 10% (0.22ml) of the total culture volume and culture is aliquoted into samples of 0.35ml that can be frozen quickly at -80ºC using dry ice.

Day 3

  1. Thaw 0.35ml of frozen competent cells quickly in a 37ºC waterbath/heating block and aliquot into 3xsterile 2.5ml micro-centrifuge,
  2. Add 0, 0.1µg and 0.5µg of the DNA to the 3 x 2.5ml tubes and incubate for 25 minutes at 37ºC in an shaking incubator at ~200rev/min
  3. After incubation add 500µl of LB medium to each of the tubes and incubate for 1-1½ hours at 37ºC with shaking to allow for the expression of antibiotic markers.
  4. Plate 0.1ml of appropriate dilutions (in LB medium) on LB agar containing selective antibiotics (Depends on transformation).
  5. Incubate the plates at 37ºC overnight and check for transformants the following day.


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Rudner Lab

Reagents

10xMC: (10ml) 1M Potassium Phosphate pH7.0 (1.36g) 30mM sodium citrate (0.088g) 20% Glucose (2g) 220mg/ml Ferric Ammonium Citrate (2200mg) 1% Casein Hydrolysate (0.1g) 2% Potassium Glutamate (0.2g)

1xMC + 3mM MgSO4: 900 μl ddH2O 100 μl 10xMC (stored at –20oC) 3 μl 1M MgSO4 (100ml stock 1M MgSO4 - 24.627g)

LB plate PstI and EcoRI Plasmid DNA (concentration not specified) Plate containing DH5α or XL1-Blue

Protocol

Day 1

  • Inoculate 3x1ml 1xMC (+3mM MgSO4) from a plate of B.subtilis in a 10ml tube and place in the shaking incubator at 37oC for 4 hours
  • Shortly before 4 hrs have elapsed prepare 5ml tubes for transformation by adding

9μl and 1μl of this solution used to transform. At the end we want 2x19μl volumes and 2x 1μl volumes and one empty control tube.

  • After 4 hrs of growth add 20-200μl of competent cells to the transformation tubes
  • Place transformation tubes back in the incubator for 37oC and shake for 2 hours
  • Plate the entire transformation on selective medium corresponding to the antibiotic resistance of the plasmid/construct. If too many transformants are yielded then use 1/20th (~50μl) of the transformation on one plate and the rest (~950μl) on a second plate.
  • Incubate overnight at 37oC
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