Team:University of Chicago/Notebook/TOP10 competent cells

TOP10 Competent Cells

 * Prechill plasticware and glassware

Preparing seed stocks

 * 1) Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C
 * 2) * room temperature works well
 * 3) Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
 * 4) * room temperature works well
 * 5) Add glycerol to 15%
 * 6) Aliquot 1 ml samples to Nunc cryotubes
 * 7) Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
 * 8) * This step may not be necessary
 * 9) Place in -80°C freezer indefinitely.

Preparing competent cells

 * 1) Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
 * 2) * This takes approximately 16 hours.
 * 3) * Controlling the temperature makes this a more reproducible process, but is not essential.
 * 4) * Room temperature will work. You can adjust this temperature somewhat to fit your schedule
 * 5) * Aim for lower, not higher OD if you can't hit this mark
 * 6) Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
 * 7) * Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
 * 8) * It is often easier to resuspend pellets by mixing before adding large amounts of buffer
 * 9) Gently resuspend in 80 ml of ice cold CCMB80 buffer
 * 10) * sometimes this is less than completely gentle. It still works.
 * 11) Incubate on ice 20 minutes
 * 12) Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
 * 13) Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells.
 * 14) Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
 * 15) Incubate on ice for 20 minutes
 * 16) Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates
 * 17) Store at -80°C indefinitely.
 * 18) * Flash freezing does not appear to be necessary
 * 19) Test competence (see below)
 * 20) Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.

Measurement of competence
Cache-Control: max-age=0
 * 1) Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)
 * 2) * This is at 10 pg/_l or 10-5 _g/_l
 * 3) * This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE
 * 4) Hold on ice 0.5 hours
 * 5) Heat shock 60 sec at 42C
 * 6) Add 250 _l SOC
 * 7) Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
 * 8) * using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
 * 9) * For our plasmids (pSB1AC3, pSPAT3) which are chloramphicoProxy-Connection: keep-alive

and tetracycline resistant, we find growing for 2 hours yields many more colonies
 * 1) * Ampicillin and kanamycin appear to do fine with 1 hour growth
 * 2) Plate 20 _l on AMP plates using sterile 3.5 mm glass beads
 * 3) * Good cells should yield around 100 - 400 colonies
 * 4) * Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
 * 5) * We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA