Team:Yale/Notebook/Protocols/

From 2012.igem.org

Preparation of Competent DH5-α cells

Materials:

• LB Buffer
• 50 mL polypropylene tubes
• TB Buffer
• DMSO
• DH5-α colonies or freshly thawed cells

TB Buffer (DH5-α competent cell buffer)

• 5mLs of 1M Hepes Buffer (final concentration 10 mM)
• 1.1 grams of CaCl₂*2H2O (final concentration 15 mM)
• 9.32 grams KCL (final concentration 250 mM)
• Bring to volume 300 mL; pH to 6.7 with 5 M KOH
• Add 5.44 grams MnCl₂*4H2O (final concentration 55 mM)
• Bring volume to 500 mL and sterile filter into sterile bottle

Protocol

• Start 5 mL overnight culture in LB from single DH5-α colony
• Innoculate 200 mL of LB with 2 mL of overnight culture and grow at 25^o C until OD = 0.5-0.55

• (Alternatively, innoculate 200 mL LB with 50 uL freshly thawed DH5-α cells and grow at 25^o C)

• Note – if you do it this way, it may take a looooong time (more than 10 hours) to read OD = 0.5

• Note from 11 August 2010: Using a single colony ON growth followed by inoculation of 200 mL with 2 mL of ON growth, it took 8.5 hours to reach OD = 0.5
• Notes from 11 August 2011: Used 50 uL of freshly thawed cells to start 50 mL ON growth. Using 2 mL of ON growth to inoculate 200 mL, it took 8h and 45 minutes to reach OD = 0.503. Using 4 mL of ON growth, the OD > 0.55 after 7h 45min; a closer monitoring of the OD at this stage could shorten the prep by at least 1 hour

• Transfer to 6 sterile, disposable ice-cold 50 mL polypropylene tubes (~35 mL per tube), Store on ice for 10 minutes
• Harvest cells by centrifugation at 2500xg for 10 minutes at 4^o C
• Pour off medium and stand upside down on paper towel for 1 minute to remove remaining medium
• Resuspend pellets by gently pipetting or swirling in 60 mL (total) of ice cold TB buffer
• Combine resuspended cells into a total of two tubes
• Centrifuge at 2500xg for 10 minutes at 4^o C to recover cells
• Pour of medium and stand upside down on a paper towel for 1 minute to remove remaining medium
• Resuspend pellets by gently pipetting or swirling in 20 mL (total) of ice cold TB buffer. Combine cells into a single tube
• Add 1.5 mL of DMSO and mix by swirling. Store cells on ice for 10 minutes
• Pipette 250 and/or 100 uL aliquots into chilled Eppendorf tubes. Make sure to close tightly and then flash freeze in liquid nitrogen and store at -80^o C
• Test for (1) contamination by plating untransformed cells on Amp plate and (2) competence with a transformation

Preparation of Competent BL21 and Origami Cells

• Thaw an aliquot of cells (without any plasmid in them) on ice
• To 200 mL of sterile LB, add 50 uL aliquot of the thawed cells: remember, this LB does not have any antibiotic in it, so work as sterile as possible (i.e. autoclave all solutions and use sterile pipettes)
• Grow cells in the shaker at 37^o C until they reach an OD = 0.3-0.4 @600nm (1 cm pathlength cell). This usually takes 3-6 hours.
• Aliquot into sterile 50 mL tubes and Spin down at 2700xg for 10 minutes at 4^o C; discard supernatant and let drain upside down on a paper towel for ~1 minute.
• Ice down sterile 100 mM CaCl₂ and 100 mM MgCl₂ solutions during centrifugation.
• Gently resuspend each pellet in 8 mL 0.1 M MgCl₂ and 2mL 0.1 M CaCl₂ and combine into 2 tubes (this should take 1-2 minutes per tube)
• Centrifuge 2700xg for 10 minutes and discard supernatant.
• Resuspend each pellet on ice in 4 mL of ice cold CaCl₂ and combine into one tube
• Add 2.7 mL sterile 60% glycerol and mix gently
• Aliquot 100 uL and/or 250 uL volumes into sterile (autoclaved), cold eppendorf tubes, freeze rapidly in liquid nitrogen and store at -80^o C
• Test for (1) contamination by plating untransformed cells on Amp plate and (2) competence with a transformation

CaS Deposition

Steps

• Add an appropriate amount of DI water to create concentrations listed below to a 1000ml beaker
• Heat the water to 90C stir at 200rpm <-seems fast
• Add substrate
• Wait 30min for substrate to stabilize <-seems long
• Add the correct quantities to create the following concentrations:
  • 0.033 M cadmium acetate
  • 1 M ammonium acetate
  • 28–30% ammonium hydroxide
  • 0.067 M thiourea
• Continue to add equal amounts of thiourea every five minutes
• Continue for 50 minutes
  • Rate is 2nm/minute
• Wash film with DI and dry in N2 environment
• Anneal???

Preparation of Bacillus subtilis competent cell

Based on "Molecular Biological Methods for Bacillus" (Harwood and Cutting, 1990)

Day 1

1. Streak out strains to be made competent on an LB or TBAB+G agar plate as a large patch and incubate overnight at 30^o C

Day 2

1. The following morning, prepare SpC medium (as below under "Solutions") fresh on the day of use
2. Scrape the cell growth off the plate(s) and use to inoculate 20 mL of fresh, pre-warmed SpC medium to give an OD₆₀₀ reading of ~0.5
3. Incubate the culture at 37^o C with vigorous aeration and take periodic OD readings (OD₆₀₀) to assess cell growth
4. While waiting for the rate of cell growth to depart from exponential (i.e. no significant change in cell density over 20-30 min), prepare SpII medium (as below under "Solutions") fresh
5. When exponential growth is no longer apparent, inoculate 200 mL of pre-warmed SpII medium with 2 mL of stationary-phase culture and continue incubation at 37^o C with slower aeration
6. After 90 minutes of such incubation, pellet the cells by centrifugation (8,000 g, 5 min) at RT#Carefully decant the supernatant into a sterile container and save (***NOTE: using freshly prepared SpII will not work)
7. Gently resuspend the cell pellet in 18 mL of the saved supernatant and add 2 mL 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 or ice/isopropanol bath and store at -70^o C

Solutions for competence induction in Bs

Prepare the following solutions fresh on the day of use (STERILE)

SpC

SpII

SpII + EGTA

Prepare SpII as usual, but substitute 4 mL EGTA (o.1 M, pH 8.0) for CaCl₂. SpII + EGTA can be frozen at -20^o C in small aliquots, although repeated freeze-thawing should be avoided

Transformation

1. Prepare SpII + EGTA or thaw a frozen aliquot
2. Thaw competent cells rapidly by immersing frozen tubes in a 37^o C water bath
3. Immediately add one volume (200 mL) to thawed cells, and mix gently
4. In a sterile test tube, add competent cells (0.2~0.5 mL) to the DNA solution (<0.1 mL) and incubate in a roller drum at 37^o C
5. Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media