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Team:Mississippi State/Making Competent Cells
From 2008.igem.org
(Difference between revisions)
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#Centrifuge 10-15min. in SS34 model, which is setting 05 for 15min at 5000rpm and +4C. | #Centrifuge 10-15min. in SS34 model, which is setting 05 for 15min at 5000rpm and +4C. | ||
#Pour out supernatant into same flask, autoclave this flask before disposal. | #Pour out supernatant into same flask, autoclave this flask before disposal. | ||
| - | #Resuspend cells in CaCl, using half previous volume: for example, if you originally had cells in 10ml LB, you would use | + | #Resuspend cells in CaCl, using half previous volume: for example, if you originally had cells in 10ml LB, you would use 5ml CaCl. |
| + | #Centrifuge again at same settings. | ||
| + | #Pour out supernatant. | ||
| + | #Resuspend in CaCl, this time using 2ml of CaCl. | ||
| + | #Store on Ice for 1hr. | ||
Latest revision as of 20:48, 17 June 2008
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- To make competent cells(XL1-Blue), do exactly as follows:
- Add 2ml LB Broth to Culture Tube.
- Add 1ul antibiotic for every 1ml LB(in this case, 2ul of Tetracycline).
- Get cell stock from -80C freezer.
- Scrape cells from top with tip of pipette.
- Add to LB Broth.
- Label (Culture name, date, iGEM)
- Put in 37C shaker overnight.
- Remove next morning.
- Get pipette gun from 1st drawer from left under lab bench closest to door.
- Get 10ml tube from lab bench opposite 1st lab bench.
- Add 25ml LB Broth to autoclaved flask(KEEP THE FOIL COVER!).
- Add 25ul Tet (in IGEM 2007 Box, -20C freezer)
- Add 250ul cells from overnight culture.
- Cover with aluminum
- Put in 37C shaker for 2hr.
- Check cell density
- Pour into centrifuge tubes.
- Centrifuge 10-15min. in SS34 model, which is setting 05 for 15min at 5000rpm and +4C.
- Pour out supernatant into same flask, autoclave this flask before disposal.
- Resuspend cells in CaCl, using half previous volume: for example, if you originally had cells in 10ml LB, you would use 5ml CaCl.
- Centrifuge again at same settings.
- Pour out supernatant.
- Resuspend in CaCl, this time using 2ml of CaCl.
- Store on Ice for 1hr.
