Joyce
Today, Digestion, DNA clean up with High Pure PCR purification kit of Roche, ligation, transformation to create transformants
with: PBAD-cI-LVA-DT, PhybB-cI-LVA-DT and PBAD-RBS-GFP-DT
Also, colony 6 of LasR-LVA (yesterday) grew on LB with ampicillin. Plasmid prep, glycerol stock and sample for sequencing were made.
Digestion:
cI-LVA-DT colony 3
2μl vector
1μl EcoR1
1μl XbaI
3μl Fast digest buffer
1μl Fast AP
22μl MQ
RBS-GFP-DT
3μl vector
1μl EcoR1
1μl XbaI
3μl Fast digest buffer
1μl Fast AP
21μl MQ
pBAD
10μl pBAD
1μl EcoR1
1μl SpeI
2μl Fast digest buffer
6μl MQ
PhybB
3μl PhybB
1μl EcoR1
1μl SpeI
2μl Fast digest buffer
13μl MQ
Incubate the samples at 37°C for 30 min.
Do a DNA purification with the PCR purification kit from Roche
Ligation:
PBAD-cI-LVA-DT colony 3
8.5μl vector
6.1μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.4μl MQ
PhybB-cI-LVA-DT colony 3
8.5μl vector
4.1μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
4.4μl MQ
PBAD-RBS-GFP-DT
8.5μl vector
6.3μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.2μl MQ
Self ligation:
8.5μl vector
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
8.5μl MQ
Ligate at room temperature for 30 min.
Transform as usual (see 12th of July)
Plasmid prep of overnight culture construct: LasR-LVA-DT (plasmid backbone with ampicillin resistance)
Elute in 100μl MQ
Use 10μl for gel electrophoresis to check wether a plasmid is present in the sample. Plasmid was seen on a 1% agarose gel with
TBE (with the help of gelred)
Nanodrop result: 67.2 ng/μl.
Send for sequencing: 5μl Sample + 5μl primer of 5μM.
Glycerol stock was made: 250μl Glycerol 85% + 750μl bacterial culture (overnight culture)
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