Difference between revisions of "Team:MIT/Protocols"

This is the media used for C. Hutch and co cultures with E. Coli. The media contains all the salts needed for both bacteria but not the carbon source (filter paper) due to the carbon source not being liquid. Cultures are made by adding the salt solution to a tube containing a piece of filter paper where the tube with the paper in it has been sterilized (usually by autoclave).

C Hutch ALONE in 1 L

Paper: 50 grams per liter of media, which is one 0.5g 9cm No. 1 filter paper disks per 100 mL.

Autoclave 15 min.

E Coli ALONE (M9) in 1 L

1. Make 5x M9 salts
   • To make M9 Salts aliquot 800ml H₂O and add
     • 64g Na₂HPO₄-7H₂O
     • 15g KH₂PO₄
     • 2.5g NaCl
     • 5.0g NH₄Cl
     • Stir until dissolved
     • Adjust to 1000ml with distilled H₂O
     • Sterilize by autoclaving
2. Measure ~700ml of distilled H₂O (sterile)
3. Add 200ml of M9 salts
4. Add 2ml of 1M MgSO₄ (sterile)
5. Add 20 ml of 20% glucose (or other carbon source)
6. Add 100ul of 1M CaCl₂ (sterile)
7. Adjust to 1000ml with distilled H₂O

C Hutch is slow to grow, so experiments will be much faster if large initial concentrations of C Hutch is used. This is achieved by having a continual stock of C Hutch in the incubator which is used to provide cells for experiments

Protocol:

Stripette:

• 10ml of current stock
• 90ml of DSM3T media
• 5g of autoclaved filter paper strips

into a 500ml flask, and place in shaker.

1. Make sure that the incubator (30/37C) and heat block (42C) are ON.
   1. Put water in the wells of the 42°C heat block.
2. Make sure required antibiotic plates are present.  Make sure you're using the right antibiotic plates for your plasmid's resistance!
   1. Warm plates to 37°C.  Cold plates reduce transformation efficiency by an order of magnitude.
   2. Also warm 500 µl SOC per transformation to room temperature (if it was in the refrigerator.)
3. Take the DNA out of --20 freezer, let it thaw.
   1. Vortex DNA to mix, then spin down.  Make sure it is completely thawed out!
4. Make sure that all of the required reagents/DNA etc are present at the site of transformation before you take the cells out of the -80.
5. Thaw the competent cells on ice for 3-4 min.
   1. You want to add your DNA right as the last bit of cells' ice melts.  Even if it's still a little slushy, that's okay.
6. Add 1-2 µl of DNA into the comp cells.  Stir with a pipette tip a few times, then put right back on ice.
   1. If you're transforming the result of a reaction (GG, LR, etc) add 1-2 µl of the reaction.  Don't add more: many of these reactions have additives that screws up transformation.
   2. If you're transforming plasmid DNA (from a miniprep), either (a) dilute it out so you add only ~10 ng of DNA, or (b) plate only 10 µl of the outgrowth – else you'll get a lawn!  Super-coiled DNA transforms super-efficiently. As an alternative, transform 1 ul of miniprep DNA and then streak the outgrowth instead of plating with beads.
   3. You can rescue DNA from an empty mini prep tube by gently pipetting the cells into the empty DNA tube, enough DNA will be stuck to the walls.
7. Incubate the cells on ice for 30-40 min.
8. Heat shock the cells for EXACTLY 30 sec at 42 C water bath.
9. Place back on ice for 90 seconds.
10. Add 250 ul of SOC (37° to RT) medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)
11. Shake the tubes at 37 C, 280 rpm for 60 min.
12. Plate 100 µl for a reaction product, or 10 µl in a 100 µl puddle of water for a supercoiled plasmid.
13. Incubate plates upside down overnight at 37 C or 16-18h at 30C.
    Can leave the cells in the incubator for up to 18 hours but no more.