Team:University of Chicago/Notebook/TOP10 competent cells

From 2008.igem.org

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
   • room temperature works well
2. Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
   • room temperature works well
3. Add glycerol to 15%
4. Aliquot 1 ml samples to Nunc cryotubes
5. Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
   • This step may not be necessary
6. 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
   • This takes approximately 16 hours.
   • Controlling the temperature makes this a more reproducible process, but is not essential.
   • Room temperature will work. You can adjust this temperature somewhat to fit your schedule
   • Aim for lower, not higher OD if you can't hit this mark
2. Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
   • Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
   • It is often easier to resuspend pellets by mixing before adding large amounts of buffer
3. Gently resuspend in 80 ml of ice cold CCMB80 buffer
   • sometimes this is less than completely gentle. It still works.
4. Incubate on ice 20 minutes
5. Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
6. Test OD of a mixture of 200 _l SOC and 50 _l of the resuspended cells.
7. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
8. Incubate on ice for 20 minutes
9. Aliquot to chilled screw top 2 ml vials or 50 _l into chilled microtiter plates
10. Store at -80°C indefinitely.
    • Flash freezing does not appear to be necessary
11. Test competence (see below)
12. 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

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

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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
   • Good cells should yield around 100 - 400 colonies
   • Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
   • We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA

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