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JULY: WEEK 2
July, 5th
LB agar plates prepared:
- LB+Amp 100 (500ml)
- LB+Cm 34 (500ml)
- LB (250ml)
- LB+Cm 12.5 (250ml)
July, 6th
We decided to perform a TECAN test to evaluate the strength of some promoters belonging to the Anderson Promoters Collection. Inoculum from glycerol stocks (8ul in 5ml LB) for:
- <partinfo>BBa_J23101</partinfo>
- <partinfo>BBa_J23110</partinfo>
- <partinfo>BBa_J23116</partinfo>
- <partinfo>BBa_J23118</partinfo>
- <partinfo>BBa_J23114</partinfo>
- <partinfo>BBa_B0033</partinfo>
TECAN experiment started at 10:20am.
In the morning one single colony was picked for BW53474 E. coli strain and glycerol stock was prepared after about 7 hours (37°C 220rpm)
Inoculum of:
- BW53474 (falcon containing culture for glycerol stock was re-filled with 5ml LB)
- MG1655 (from glycerol stock)
- BW25141 (from glycerol stock)
- BW25142 (from glycerol stock)
July, 7th
Competent cells preparation for:
- MG1655
- BW53474
- BW25141
- BW25142
PCR was performed to amplify Phasins:
All parts were correct!! :)
July, 8th
Transformation of 1ul (~4ng) of miniprepped <partinfo>BBa_J23118</partinfo> into 100ul of home-made competent cells
- MG1655
- BW25141
- BW25142
- BW53474
- DH5-alpha (as control)
to verify the transformation efficiency.
Transformed bacteria were plated on LB+Amp and left overnight in oven, 37°C.
July, 9th
We checked the presence of colonies in plates.
All plates showed red colonies!
 MG1655 plate (with satellite colonies) | |
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We calculated (thanks Nicolò) efficiency as #colonies/ug DNA
| Strain | #colonies | Efficiency
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| MG1655 | 379 | ~10^5
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| BW25141 | 1431 | ~10^5
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| BW25142 | 2374 | ~10^5
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| BW53474 | 9984 | ~10^6
(very difficult to count correctly)
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| DH5alpha | 7475
(only 100ul of cells plated) | ~ 10^6
(very difficult to count correctly,
previous tests showed an efficiency of ~10^8)
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