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Team:Osaka/Protocols
From 2011.igem.org
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== Protocols == | == Protocols == | ||
=== Cell survival assay === | === Cell survival assay === | ||
| - | #Pre-culture transformed cells in 3ml of LB medium at 37°C for | + | #Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h. |
| + | #IPTGを終濃度が100μMになるまで加えて、37℃一時間 | ||
#Dilute pre-culture samples according to intended UV energy dosage (if dosage is high, lower dilution rate is needed as fewer cells are expected to survive). | #Dilute pre-culture samples according to intended UV energy dosage (if dosage is high, lower dilution rate is needed as fewer cells are expected to survive). | ||
#Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness. | #Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness. | ||
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#Wrap plates in aluminium foil and incubate at 37°C. | #Wrap plates in aluminium foil and incubate at 37°C. | ||
#After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates. | #After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates. | ||
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Revision as of 22:40, 26 October 2011
Protocols
Cell survival assay
- Pre-culture transformed cells in 3ml of LB medium at 37°C for 16h.
- IPTGを終濃度が100μMになるまで加えて、37℃一時間
- Dilute pre-culture samples according to intended UV energy dosage (if dosage is high, lower dilution rate is needed as fewer cells are expected to survive).
- Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.
- Irradiate cells on the agar with UV light at desired energy dosage.
- Wrap plates in aluminium foil and incubate at 37°C.
- After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.
SOS Promoter assay
- Pre-culture transformed cells in 8ml of LB medium at 37°C for 12h.
- Transfer pre-culture to OD600 measurement dish (ø50mm).
- Irradiate with UV light at desired energy dosage.
- Incubate irradiated cells for a further 2h.
- Measure OD600 as a measure of cell density.
- Transfer cells into 15ml Falcon tubes and centrifuge.
- Discard supernatant, add 1ml water to wash cells.
- Repeat centrifugation and decantation.
- Add 500μl acetone and vortex.
- Incubate at 55°C for 15min.
- Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 measurements.
