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Team:Hawaii/Notebook/2008-06- 2
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* Tested OD600 every hour for 90 mL SOB mixture and 250 mL SOB mixture made yesterday | * Tested OD600 every hour for 90 mL SOB mixture and 250 mL SOB mixture made yesterday | ||
:* Growth until OD = 0.3 - 0.6, cells then made competant | :* Growth until OD = 0.3 - 0.6, cells then made competant | ||
| - | :* OD600 is ideal for measuring Mach10 growth; OD550 should be used for DH5-alpha (citation?) | + | :* OD600 is ideal for measuring Mach10 growth; OD550 should be used for DH5-&alpha (citation?) |
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| + | ==== Growth of DH5-&alpha from seed stock==== | ||
| + | Added one 0.9 mL seed stock to 90 mL SOB and checked growth (OD550 & OD600) every hour | ||
| + | |||
====Competent Cells==== | ====Competent Cells==== | ||
* Competent cells prepared according to [[Team:Hawaii/Make_Competent_E._Coli | protocol]] based on Hanahan | * Competent cells prepared according to [[Team:Hawaii/Make_Competent_E._Coli | protocol]] based on Hanahan | ||
Revision as of 04:30, 3 June 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Contents |
Made Competent Cells
Grace, Margaret, Norman
Seed Stock
- Tested OD600 of 1 seed stock tube @ 16 hr after innoculation
- OD600 = 0.53244
- Separated each 1.8 mL seed stock tube to two 0.9 mL tubes
- Original cryotubes = 2 mL, which is not big enough for addition of glycerol later
- Next time, perhaps use other type of tube (cryotubes don't allow air for E. coli to grow)
- Incubated at 37C with shaking for additional 3 hrs
- Protocol suggests 23C but growth was too slow. Also, protocol is for Mach10 cells, which grows much faster.
- Added 158.8 ul glycerol to 6 seed stock tubes per compentency protocol
- Added 327.8 ul glycerol to 15 seed stock tubes (26.7% glycerol) per SC's recommendation
Growth curve
- Took one 0.9 mL seed stock tube prior to additional incubation and added to 90 mL SOB media
- Tested OD600 every hour for 90 mL SOB mixture and 250 mL SOB mixture made yesterday
- Growth until OD = 0.3 - 0.6, cells then made competant
- OD600 is ideal for measuring Mach10 growth; OD550 should be used for DH5-&alpha (citation?)
| Time | OD for 90 mL solution | OD for 250 mL solution |
|---|---|---|
| 11AM | OD600 = 0.007 | OD600 = 0.12 |
| 12PM | OD600 = 0.01089 | OD600 = 0.22 |
| 1PM | OD600 = 0.05371 | OD600 = 0.48099, OD550 = 0.5303 |
| 2PM | OD600 = 0.15752, OD550 = 0.17463 | OD600 = 0.57673, OD550 = 0.63531 |
| 3PM | OD600 = 0.33753, OD550 = 0.37611 | n/a |
Growth of DH5-&alpha from seed stock
Added one 0.9 mL seed stock to 90 mL SOB and checked growth (OD550 & OD600) every hour
Competent Cells
- Competent cells prepared according to protocol based on Hanahan
- Competent cells were made from stocks w/ OD600 = 0.33753 and 0.57673
- 250 ul chilled CCMB80 was added to each 50 ul aliquot of OD600=~0.6 to get final OD600 = 1.0-1.5
- 100 ul competent cells were aliquoted into each tube and stored at -80C
| Tube | OD600 | OD550 |
|---|---|---|
| 1 | 1.1936 | 1.2488 |
| 2 | 1.1182 | 1.1718 |
| 3 | 1.1289 | 1.1849 |
| 4 | 1.0059 | 1.0679 |
Note: Desired OD600 = 1.0-1.5
Discussion
Transform E.coli with PRL1383a tomorrow?
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
