Team:Uppsala-Sweden/Notebook
From 2011.igem.org
(→Week 2) |
(→Week 3) |
||
Line 136: | Line 136: | ||
'''2011-07-04''' | '''2011-07-04''' | ||
- | + | Started by doing overnight cultures of the strains carrying these plasmids: | |
+ | |||
+ | pSB1A3-J04450 (vector, ampR) | ||
+ | pSB1C3-J04450 (vector, CmR) | ||
+ | pSB1K3-J04450 (vector, KanR) | ||
+ | pSB1A2-B0034 (Standard RBS) | ||
+ | pSB2K3-I15008 (ho1, chromophore gene) | ||
+ | pSB2K3-I15009 (pcyA, chomophore) | ||
+ | pSB2K3-Q03530 (cII inverter) | ||
+ | pSB1AK3-B0015 (Double terminator) | ||
+ | pSB2K3-Q01511 (cI inverter) | ||
+ | pSB1A2-R0082 (PompC) | ||
+ | pSB2K3-I15010 (cph8 red sensor) | ||
+ | |||
+ | The purpose was to make plasmid preparation the day after. | ||
+ | |||
'''2011-07-05''' | '''2011-07-05''' | ||
- | + | ||
+ | |||
+ | The day started of by running a PCR on the DNA template of the green light sensor which includes the two genes ccaR and ccaS as well as the promotor PcpcG2 (see protocol ccaR BioBrick, protocol ccaS BioBrick, protocol PcpcG2 BioBrick). | ||
+ | |||
+ | After lunch we did a plasmid preparation of the overnight cultures from 4/7 (purification protocol). | ||
+ | At the end of the day we did a PCR to verify the red light sensor (cph8). We also did site specific mutagensis using PCR for the pigments amilGFP and amilCP in order to remove illegal EcoRI sites within the genes (protocol cph8, protocol amilGFP_EcoRI, protocol amilCP_EcoRI) | ||
+ | |||
Revision as of 13:15, 7 July 2011
Uppsala University.
Welcome to Uppsala-SwedeniEM '2011
This is the looong logo
Notebook
|
|
|
|
|
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.
2011-07-01
All the overnight cultures prepared 11-07-01 were frozen in -80°C.
Week 3
2011-07-04
Started by doing overnight cultures of the strains carrying these plasmids:
pSB1A3-J04450 (vector, ampR) pSB1C3-J04450 (vector, CmR) pSB1K3-J04450 (vector, KanR) pSB1A2-B0034 (Standard RBS) pSB2K3-I15008 (ho1, chromophore gene) pSB2K3-I15009 (pcyA, chomophore) pSB2K3-Q03530 (cII inverter) pSB1AK3-B0015 (Double terminator) pSB2K3-Q01511 (cI inverter) pSB1A2-R0082 (PompC) pSB2K3-I15010 (cph8 red sensor)
The purpose was to make plasmid preparation the day after.
2011-07-05
The day started of by running a PCR on the DNA template of the green light sensor which includes the two genes ccaR and ccaS as well as the promotor PcpcG2 (see protocol ccaR BioBrick, protocol ccaS BioBrick, protocol PcpcG2 BioBrick).
After lunch we did a plasmid preparation of the overnight cultures from 4/7 (purification protocol). At the end of the day we did a PCR to verify the red light sensor (cph8). We also did site specific mutagensis using PCR for the pigments amilGFP and amilCP in order to remove illegal EcoRI sites within the genes (protocol cph8, protocol amilGFP_EcoRI, protocol amilCP_EcoRI)
2011-07-06
Placeholder.
2011-07-07
Placeholder.
2011-07-08
Placeholder.
Week 4
2011-07-11
Placeholder.
2011-07-12
Placeholder.
2011-07-13
Placeholder.
2011-07-14
Placeholder.
2011-07-15
Placeholder.
Week 5
2011-07-18
Placeholder.
2011-07-19
Placeholder.
2011-07-20
Placeholder.
2011-07-21
Placeholder.
2011-07-22