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Team:Groningen/project notebook/11 August 2011
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| + | =Thu 11 August 2011= | ||
| + | '''Vessa''' | ||
| + | ==To Do List== | ||
| + | *Plasmid prep and send for sequencing | ||
| + | **12 variants autoinducing loop | ||
| + | **RFP internal control | ||
| + | **construct 21.12 | ||
| + | *Pick colonies of back up transformation ---Growth ON culture | ||
| + | |||
| + | ===Implementation=== | ||
| + | '''Result 1st batch transformation''' | ||
| + | <br>all of 22.xx and 21.12 plasmids have no insert | ||
| + | |||
| + | RBS-RFP PCR colonies screening --> pick 8 colonies --> ALL POSITIVE! *\(^___^)/* Hoera!! -> isolate the plasmids and send for sequencing! | ||
| + | |||
| + | |||
| + | '''Highlight''' | ||
| + | *Weekly team meeting at 1 pm | ||
| + | *Modelling lecturer from Prof. Matthias Heinemaann | ||
'''Joyce''' | '''Joyce''' | ||
<br> | <br> | ||
Latest revision as of 11:55, 21 September 2011
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Thu 11 August 2011
Vessa
To Do List
- Plasmid prep and send for sequencing
- 12 variants autoinducing loop
- RFP internal control
- construct 21.12
- Pick colonies of back up transformation ---Growth ON culture
Implementation
Result 1st batch transformation
all of 22.xx and 21.12 plasmids have no insert
RBS-RFP PCR colonies screening --> pick 8 colonies --> ALL POSITIVE! *\(^___^)/* Hoera!! -> isolate the plasmids and send for sequencing!
Highlight
- Weekly team meeting at 1 pm
- Modelling lecturer from Prof. Matthias Heinemaann
Joyce
Plasmid prep of overnight culture PBAD-pSB1C3 colonies 1 and 2
Measure DNA concentration: 32 ng/microliter
Samples were send for sequencing and glycerol stocks were made
colony PCR of PBAD-pSB1C3:
Composition master mix:
10× Taq buffer: 40μl
dNTPs 10mM: 8μl
MgCl2: 24μl
Forward biobrickvector primer: 8μl
Reverse biobrickvector primer: 8μl
Taq polymerase: 2μl
MQ water: 310μl
PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 60°C for 30s
Extenstion: 72°C for 30s
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel
dsDNA was digested:
for taRNA fragment:
5μl dsDNA plasmid
1μl SacI
1μl BamHI
2μl FD buffer
11μl MQ
dsDNA was digested:
for crRNA fragment:
5μl dsDNA plasmid
1μl BamHI
1μl SalI
2μl FD buffer
11μl MQ
dsDNA was digested:
for PRM fragment:
5μl dsDNA plasmid
1μl SalI
1μl HindIII
2μl FD buffer
11μl MQ
Isolate from 1% agarose gel with the agarose extraction kit
PCR overnight for taRNA fragment:
10× taq buffer: 5μl
dNTPs: 1μl
F3: 1μl
R4: 1μl
pfu: 0.04μl
taq: 0.25μl
DNA template: 5μl
33.71μl MQ
PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (35×)
Denaturation: 94°C for 30s
Annealing: 53°C for 30s
Extenstion: 72°C for 1min
Final extension: 72°C for 10min
Store infinite at 4°C
