Jori
GA algorithm and database crossover
Vessa
Troubleshooting
Cloning:
- Overdigestion
- Vector and insert mass are TOO low
- Vector insert ratio is TOO low
Next step to do
- Plasmid prep of Pc and ES digestion
- Doubled the concentration of ligation mix
- Increase the vector-insert ratio =1:9
- Elute the digested vector in 25 ul!
- Prepare 10 reaction tubes of insert digestion --> clean up in 2 tubes
Highlight
Meeting with Dr. Juke Lolkema
Joyce
Plasmid prep of taRNA-pSB1A3-DT and RBS-GFP-DT (again)
DNA concentrations were around 30ng/μl. De taRNA-pSB1A3-DT were send for sequencing and glycerol stocks were made.
Plasmidpreps of RBS-GFP-DT and taRNA-pSB1A3-DT (1 and 4) were used for vector
Checked sequencing results PBAD/araC-pSB1C3, were alright!:)
Digestion:
RBS-GFP-DT
5μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl FastAP
19μl MQ
taRNAc1-pSB1A3-DT
7.5μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl FastAP
16.5μl MQ
taRNAc1-pSB1A3-DT
5μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl FastAP
19μl MQ
PBAD
5μl insert
1μl EcoRI
1μl SpeI
2μl Fast digest buffer
11μl MQ
PhybB
4μl insert
1μl EcoRI
1μl SpeI
2μl Fast digest buffer
12μl MQ
Incubate for 1 hour at 37 degrees
After incubation: DNA clean up with PCR purification kit of Roche
Ligation:
PBAD/araC-RBS-GFP-DT
8.5 μl vector
5.7 μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.8μl MQ
PhybB-taRNA-pSB1A3-DT
8.5 μl vector
6.5 μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2μl MQ
Self ligation control:
8.5 μl vector
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
8.5μl MQ
incubate the samples for 40 minutes at room temperature
For transformation: take a sample of the plasmid PhybB-RBS-GFP-DT (5μl of the plasmid) and transform this in TOP10 competent
cells in parallel with the other samples. This will be done because of the DH5alpha strain does not grow well in M9 medium for
flow cytometry measurements...
Transformation:
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight
Also, in the morning, the plate with PhybB-RBS-GFP looked alright, had single colonies, so I let them grow this morning in 30
degrees for induction of the PhybB promotor. After 1 and 3 hours, no fluorescent colonies were seen on the plate.
"
