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==PCR==
==PCR==


#This protocol is desgined for use with the stratagene ''PfuUltra'' II Fusion DNA polymerase and is according to the DNA polymerase usage manual. [http://www.stratagene.com/manuals/600670.pdf ''PfuUltra'' II Fusion Manual]
This protocol is desgined for use with the stratagene ''PfuUltra'' II Fusion DNA polymerase and is according to the DNA polymerase usage manual. [http://www.stratagene.com/manuals/600670.pdf ''PfuUltra'' II Fusion Manual]


=====Aim=====
=====Aim=====

Revision as of 04:51, 6 August 2008

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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

Preparation of competent B. subtilis cells and transformation with plasmid DNA – the ‘Paris method’

Media

MMx5: Identical to minimal salts (x5) from Groningen method except that MgSO4 is omitted.

MG: 20ml MMx5; 2.5ml glucose (20% (w/v)); 0.5ml MgCl2 (1M); H2O to 100ml.

MG1: 50ml MG; 0.5ml casamino acids (2% (w/v)); amino acid growth factors to 50µg/ml each.

MG2: 100ml MG; 0.5ml casamino acids (2% (w/v)); amino acid growth factors to 10µg/ml each.

Transformation buffer: 2ml MMx5; 0.2ml MgCl2 (1M); 0.25ml glucose (20% (w/v)); 0.1ml EGTA (0.1M); H2O to 10ml.

Competent cells
  1. Inoculate TBAB agar slant in a test tube (surface of the agar ~10cm2) with a fresh culture. Incubate overnight at 30ºC (allow for sufficient oxygen supply by not tightening the screw cap).
  2. Resuspend cells from the TBAB slant into 5ml of medium MG1 in a 250ml Erlenmeyer flask. Adjust the OD650 to ~0.7.
  3. Incubate the culture for 3 hours at 37ºC in a waterbath shaker (200rev/min).
  4. Dilute the culture 10-fold in 50ml of pre-warmed MG2 in a 1-1 Erlenmeyer flask.
  5. Incubate for 90 minutes with vigorous shaking at 37ºC.
  6. Pellet the cells by centrifugation (5000g, 5 minutes, 20ºC; pre-warm rotor). Save the supernatant as well; add to it glycerol to 8.5% (v/v) and glucose to 0.5%. Mix.
  7. Immerse the microfuge tubes in a liquid nitrogen bath.
  8. Resuspend the cell pellet gently in 4.5ml of the supernatant (plus glycerol and glucose). Mix gently.
  9. Distribute competent cells in small portions in microfuge tubes (placed in liquid nitrogen) and transfer these quickly to -80ºC.
Transformation
  1. Add 1-10µl of DNA solution to a 1.5ml microfuge tube.
  2. Thaw quickly (37ºC, waterbath) an aliquot of frozen competent cells and dilute this 10-fold in transformation buffer. Mix gently (do not vortex).
  3. Transfer 100µl of thawed cells to the DNA in the microfuge tube, mix and incubate for 20 minutes at 37ºC (without shaking).
  4. Add 500µl of LB medium and incubate for 1-1½ hours at 37ºC with shaking to allow for the expression of antibiotic markers.
  5. Plate on LB agar plates supplemented with appropriate antibiotics.

Preparation of competent B. subtilis cells and transformation with plasmid DNA – the ‘Groningen method’

Media

Minimal salts (x5), per litre: (NH4)2SO4, 10g; K2HPO4, 74g; KH2PO4, 27g; trisodium citrate, 9.5g; MgSO4.7H2O, 1.0g; pH7.0. Glucose: 20% (w/v) solution. Casamino acids: 2% (w/v) solution. Minimal-growth medium per 100ml: 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). Starvation medium per 100ml: 20ml minimal salts; 2.5ml glucose.

Competent cells
  1. Plate the appropriate strain to check the phenotype (auxotrophic markers, antibiotic resistances etc.).
  2. Inoculate 10ml of minimal growth medium in a 100ml flask with cells from a single colony.
  3. Grow overnight (~16-18 hours) with agitation (waterbath shaker, 200rev/min).
  4. Add 1.4ml of overnight culture to 10ml of pre-warmed fresh minimal-growth medium (plus growth factors) in a 100ml flask.
  5. Grow for 3 hours at 37ºC with agitation (shaker, 200rev/min).
  6. Add 11ml of starvation medium and continue growth for 2 hours at 37ºC with vigorous agitation (shaker, 300rev/min).
  7. The culture is now maximally competent (for at least ½ an hour).
  8. Freeze the culture for future use. Add sterile glycerol to 10% (v/v), mix and disperse in small portions (e.g. 0.5ml). Freeze quickly at -80ºC or in liquid nitrogen. The loss of competency is usually less than two-fold. Use a fresh aliquot each time.
Transformation
  1. Transfer 100µl of freshly prepared competent cells to a sterile 2.5ml micro-centrifuge tube (or thaw quickly in a 37ºC waterbath an aliquot of frozen competent cells and use immediately).
  2. Add 1-10µl of plasmid DNA and mix. For saturating amounts of DNA, add 0.2-0.5µg instead.
  3. Incubate for 25 minutes at 37ºC in a waterbath shaker (200rev/min).
  4. Add 500µl of LB medium and incubate for 1-1½ hours at 37ºC with shaking to allow for the expression of antibiotic markers.
  5. Plate 0.1ml of appropriate dilutions (in LB medium) on LB agar containing selective antibiotics.
  6. Incubate the plates at 37ºC.

One-step Transformation Procedure

  1. Streak recipient strain heavily on one-half of a TBAB agar plate. Incubate for 18 hours at 37ºC.
  2. Inoculate into (n + 0.5)ml of MB in a test tube (where n is the number of selective plates you will use) heavily enough so that slight turbidity is visible. Incubate with aeration for 2 hours.
  3. Distribute 1ml of the competent culture into n labelled tubes. Add 0.1ml or less of sterile DNA to each tube and incubate for 1 hour.
  4. Add 2.5ml of SC to each tube. Centrifuge (8000g, 10 minutes, RT) and discard supernatant.
  5. Resuspend each cell pellet in 0.2ml of SC. Plate 0.1ml of undiluted suspension on selective plates.


Two-step Transformation Procedure

Preparation of competent cells
  1. Streak out the strain to be made competent on an LB or TBAB agar plate as a large patch and incubate overnight at 30ºC.
  2. The following morning, scrape the cell growth off the plate and use to inoculate fresh, pre-warmed SpC medium (20ml) to give an OD600 reading of about 0.5.
  3. Incubate the culture at 37ºC with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.
  4. When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 minutes) inoculate 200ml of pre-warmed SpII medium with 2ml of stationary-phase culture and continue incubation at 37ºC with slower aeration.
  5. After 90 minutes incubation, pellet the cells by centrifugation (8000g, 5 minutes) at room temperature.
  6. Carefully decant the supernatant into a sterile container and save.
  7. Gently resuspend the cell pellet in 18ml of the saved supernatant and add 2ml of sterile glycerol; mix gently.
  8. Aliquot the competent cells (0.5ml) in sterile tubes, freeze rapidly in liquid nitrogen or a dry-ice/ethanol bath and store at -70ºC.
Transformation
  1. Thaw competent cells rapidly by immersing frozen tubes in a 37ºC water bath,
  2. Immediately, add one volume of SpII + EGTA to the thawed cells; mix gently.
  3. In a sterile test tube add competent cells (0.2-0.5ml) to the DNA solution (<0.1ml) and incubate in a roller drum at 37ºC.
  4. Dilute the transformed cells as appropriate in T Base containing 0.5% glucose and plate immediately onto selective media.

Motility Experiments

Isolating motile derivatives

  1. Streak each strain carefully at one point of a 0.7% TBAB agar plate.
  2. Incubate overnight at 37ºC without inverting the plates.
  3. Motile cells will swarm over the surface of the plate and should be picked from as far from the site of inoculation as possible. If the strain was non-motile there will be a contained streak with a few dendritic swarms emerging from the streak. Pick from the top of one of these swarms to recover a motile derivative.


PCR

This protocol is desgined for use with the stratagene PfuUltra II Fusion DNA polymerase and is according to the DNA polymerase usage manual. PfuUltra II Fusion Manual

Aim

To produce clones of two genes from B.subtilis that are too big to have synthesised by GeneArt; for use as integration sequences (AmyE and PyrT) or for their original purpose (XylR).

Equipment

Heated lid PCR machine

Thin walled PCR tube

Reagents
Reagent Volume
Distilled H2O 16μL
10Χ PfuUltra®II reaction buffer 5μL
dNTP mix (12.5mM each dNTP) 0.5μL
B.subtilis genomic DNA (50ng/μL) 1μL
Forward Primer (5μM) 1μL
Reverse Primer (5μM) 1μL
PfuUltra® II fusion HS DNA polymerase 0.5μL
Total Reaction Volume 25μL

Note. Template DNA should be diluted to 100ng/μL. If template DNA concentration is below 100ng/μL, 50ng of DNA should be added and the volume of H2O to be added should adjusted to maintain a reaction volume of 25μL

The forward and reverse primers should contain the Biobrick prefix (forward primer) and the complementary sequence to the Biobrick suffix (reverse primer) 5' of the beginning of the annealing sequence

Protocol

Add all the reagents in order (down the list) sequentially to the PCR tube mxing after each addition. Place PCR tubes into th ePCR machine and set the programme to the following set-up:

Initial Denaturation: 2 minutes at 95°C
30 Cycles of: 20 second denaturation at 95°C
20 second annealing time at Primer Tm - 5°C
15 second extending time at 72°C
Final Extension: 3 minutes at 72°C

The resulting solution can then be purified using a PCR purification column or by gel electrophoresis followed by spin purification.


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