Team:Uppsala-Sweden/Notebook
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Uppsala University.
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Notebook
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Week 1
This week was dedicated to make buffers such as CCMB80 and SOC, SOB and LB medium (See protocol SOB- medium, LB medium and Competent cell preparation final). We also prepared selective agar plates with ampicillin, chloramphenicol and kanamycin (See agarplate preparation). Finally we prepared competent TOP10 cells and froze them in -80°C (See Competent cell preparation final).
Week 2
2011-06-27
- This week we started with testing the competence in our cells. For this transformation we used one positive control (plasmid pUC19 from New England Biolabs) as well as a negative control without plasmid. For the procedure we followed the protocol for transforming TOP10 competent cells. The result was good, we measured a competence efficiency of 1.7 * 108 transformants /ug DNA.
- Started overnight cultures of E coli carrying the plasmids pGEM11- amilGFP (green output) and pGEM14- amilCP (blue output). For this procedure we followed the protocol Overnight culture and glycerol stock. The strains carrying the amilGFP and amilCP plasmids were provided by J.F Miller, UCLA.
2011-06-28
- Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):
pSB1A3-J04450 (ampR backbone) pSB1C3-J04450 (CmR backbone) pSB1K3-J04450 (KanR backbone) pSB1AK3-B0014 (Double terminator) pSB1AK3-B1001 (synthetic terminator) pSB1A2-B0034 (Standard RBS) pSB2K3-I15008 (ho1, chromophore gene) pSB2K3-I15009 (pcyA, chomophore) pSB1A2-R0011 (PllacO, lacI repressable promotor) pSB2K3-I15010 (cph8 red sensor)
2011-06-29
- Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):
pSB2K3-Q03530 (cII inverter) pSB3T5-J04450 (low copy vector tetR, ori P15A) pSB1AK3-B0015 (Double terminator) pSB2K3-Q01511 (cI inverter) pSB1A2-R0082 (PompC) pSB1A2-K093005 (RBS + RFP, red dye) pSB2K3-I15010 (cph8 red sensor) - had to be redone due to failure in the first attempt.
Followed up by restreaking of the transformants from the previous day.
2011-06-30
- Restreaking of the transformants from 11-06-29 and started overnight cultures of the reastreaked plates from the previous day (11-06-28).
2011-07-01
- Started with doing overnight cultures from the reastreaked plates from the previous day (11-06-30).
After that we prepared 20 % sterile glycerol for making glycerol stocks. All the overnight cultures prepared 11-06-30 were frozen in -80°C.
Week 3
2011-07-04
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2011-07-05
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2011-07-06
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2011-07-07
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2011-07-08
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Week 4
2011-07-11
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2011-07-12
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2011-07-13
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2011-07-14
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2011-07-15
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Week 5
2011-07-18
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2011-07-19
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2011-07-20
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2011-07-21
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2011-07-22