User:Andriana/5 June 2012

From 2012.igem.org

I. Plated DH5-α cells on plain agar plate (making competent DH5-α cells)

Note: Instead of plating, growth in a culture flask can be begun directly

1. Have a plain agar plate on hand
2. Sterilize an inoculating loop by spraying it with an alcohol like ethanol right before heating it with a Bunsen burner until it glows orange-red
3. Use the inoculating loop to scrape some frozen (NOT THAWED) DH5-α cells from an aliquot and spread them around the outer radius of the agar plate making haphazard lines with sharp angles.
4. Let the bacteria grow overnight in a 30C environment. Colonies should grow in the center of the plate, but bacteria on the outside may also be used if this does not happen.

II. Make TB Buffer (in this order):

1. Dissolve 1.1915 g HEPES in 5 mL of DI water
2. Measure 1.1 g CaCl₂ X 2H₂O# 9.32 g KCl
3. Bring volume to 300 mL
4. Using a pH probe, bring volume to pH 6.7 using stock of 5 M KOH. Careful!: Once pH is about 5.5, dilute your stock with some DI water and use a pipette to bring pH to desired level because pH may spike very quickly.
5. Add 5.44 g MnCl₂ X 4 H₂O, and bring volume of solution to 500 mL# Pass the solution through sterilizing filter into a sterile container.

III. Grew and Prepared Competent Origami Cells

1. Add a thawed aliquot of origami cells to a sterile 2 L flask (thin glass non-filter flask with grooves on bottom and no lip) containing 200 mL of sterile LB broth, making sure to leave a few mL of the broth before addition of bacteria for next steps.
2. Fill a small tub, placing a tube rack inside and the solutions of MgCl₂, CaCl₂, and 60% glycerol to chill. This step should be done early enough to allow solutions to chill, but not so that all ice melts, since growing the cells will take at least 6 hours for -80C frozen aliquots. Also, chill sterile eppendorf tubes and a rack. To maintain tube sterility, the tubes should stay in their original container up until the transfer of competent cells
3. Grow this solution in 37C shaker until OD measured by a spectrophotometer is .3-.4 (about 6 hours). Use the saved aseptic LB saved in step 1 to use as a blank for this.
4. When the desired OD is reached, transfer the cell-broth solution into 4 corning tubes
5. Aliquot cells into 4 sterile 50 mL falcon tubes, each containing about the same amount of liquid and spin at 2700xg for 10 min at 4C
6. Discard the supernatant after centrifugation, keeping the pellets and resulting solutions on ice.
7. Gently resuspend the pellets in solution of 8 mL .1M MgCl₂ and 2 mL .1 M CaCl₂
8. Combine the solutions into 2 tubes and follow the second part of step 5 and step 6 again
9. Gently resuspend the pellets in 4 mL CaCl₂ each and combine the solutions into one tube
10. Add 2.7 mL sterile 60% glycerol to the tube (keep everything on ice)
11. Transfer cell-solution to eppendorf tubes at desired volume (100 or 250 microliters), and freeze in liquid nitrogen + transfer to an 80C freezer as quickly as possible. Store cells at -80C indefinitely

NOTE: All sampling for the OD and other procedures should be done aseptically near a burning Bunsen burner. Whenever cell broth is poured in our out, sterilize lips of all containers by passing them over burning flame.