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Team:Brown/Notebook/August2
From 2010.igem.org
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(New page: {{:Team:Brown/templates/header}} ==Monday, August 2, 2010== ===(Re?)digestion of RBS34===) |
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===(Re?)digestion of RBS34=== | ===(Re?)digestion of RBS34=== | ||
| + | 50ul total volume | ||
| + | *5ul NEB 10x buffer 2 | ||
| + | *39.5ul dH2O | ||
| + | *1ul EcoRI | ||
| + | *1ul SpeI | ||
| + | *0.5ul 100x BSA | ||
| + | *3ul miniprep of RBS34 | ||
| + | |||
| + | Gel appears to be cloudy and contaminated | ||
| + | |||
| + | [[Image:Gel_8210_1.jpg]] | ||
| + | |||
| + | ===Kan/Tet plate test results=== | ||
| + | None of the hand-contamination plates grew. XL1s on tet plates grew, but not BL21s. From this, we conclude that the XL1Bs may be contaminated with Tet resistance. | ||
| + | |||
| + | ===PCR of reporter system=== | ||
| + | '''Rehydration of primers''' | ||
| + | *Fwd - 156ul dH2O added | ||
| + | *Rev - 171.2ul dH2O added | ||
| + | |||
| + | '''PCR''' | ||
| + | |||
| + | 50ul total volume | ||
| + | *2ul fwd primer (10uM) | ||
| + | *2ul reverse primer (10uM) | ||
| + | *1ul template DNA | ||
| + | *25ul master mix | ||
| + | *20ul dH2O | ||
| + | |||
| + | ===Transformation of reporter constructs=== | ||
| + | *LacI reporter - from iGEM HQ, part J04421 miniprep product | ||
| + | *AraC reporter - from ligation on 7/29 of pAraC and J06702 | ||
| + | 3ul of each DNA in XL1Bs, standard 30min incubation and 1min heatshock. | ||
| + | |||
| + | - | ||
| + | |||
| + | Transformation of AraC and bistable partin DH5alpha on Kan plates, 50ug/mL | ||
Latest revision as of 19:41, 4 October 2010
Monday, August 2, 2010
(Re?)digestion of RBS34
50ul total volume
- 5ul NEB 10x buffer 2
- 39.5ul dH2O
- 1ul EcoRI
- 1ul SpeI
- 0.5ul 100x BSA
- 3ul miniprep of RBS34
Gel appears to be cloudy and contaminated
Kan/Tet plate test results
None of the hand-contamination plates grew. XL1s on tet plates grew, but not BL21s. From this, we conclude that the XL1Bs may be contaminated with Tet resistance.
PCR of reporter system
Rehydration of primers
- Fwd - 156ul dH2O added
- Rev - 171.2ul dH2O added
PCR
50ul total volume
- 2ul fwd primer (10uM)
- 2ul reverse primer (10uM)
- 1ul template DNA
- 25ul master mix
- 20ul dH2O
Transformation of reporter constructs
- LacI reporter - from iGEM HQ, part J04421 miniprep product
- AraC reporter - from ligation on 7/29 of pAraC and J06702
3ul of each DNA in XL1Bs, standard 30min incubation and 1min heatshock.
-
Transformation of AraC and bistable partin DH5alpha on Kan plates, 50ug/mL
