IGEM:Groningen/Notebook/iGEM 2011/2011/07/11

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11-7-11


PCR clean up of the pBAD araC PCR products.
Digestion of pBAD araC, cI-LVA, LasR-LVA and the vectors pSB1A3+DT and pSB1A3 + RBS-GFP
Digestion
pBAD araC:
pBADaraC: 10μl
EcoRI: 1μl
SpeI: 1μl
Fast digest buffer: 2μl
MilliQ water: 6μl

cI-LVA:
cI-LVA: 2μl
EcoRI: 1μl
SpeI: 1μl
Fast digest buffer: 2μl
MilliQ water: 14μl

LasR-LVA:
LasR-LVA: 3μl
EcoRI: 1μl
SpeI: 1μl
Fast digest buffer: 2μl
MilliQ water: 13μl

pSB1A3+DT:
pSB1A3+DT: 3μl
EcoRI: 1μl
SpeI: 1μl --> XbaI?
Fast digest buffer: 2μl
Fast alkaline phosphatase: 1μl
MilliQ water: 22μl

pSB1A3+RBS-GFP:
pSB1A3+RBS-GFP: 3μl
EcoRI: 1μl
SpeI: 1μl --> XbaI?
Fast digest buffer: 2μl
Fast alkaline phosphatase: 1μl
MilliQ water: 22μl

Incubate at 37 °C for 30 minutes
Do after the digestion a DNA clean up with the High Pure PCR purification kit --> how much MQ did you use for DNA elution?
Ligation
Calculate with the ligation calculator how much ng DNA is needed for your ligation and then calculate how many microliters this is.
cI-LVA in pSB1A3-DT
cI-LVA: 7.8 μl
pSB1A3-DT: 8.5μl
T4 DNA ligase buffer: 2μl
T4 DNA ligase: 1μl
MilliQ water: 0.7μl

LasR-LVA in pSB1A3-DT
LasR-LVA: 6.1 μl
pSB1A3-DT: 8.5μl
T4 DNA ligase buffer: 2μl
T4 DNA ligase: 1μl
MilliQ water: 2.4μl

pBAD araC in pSB1A3+RBS-GFP
pBAD araC: 7.8 μl
pSB1A3+ RBS-GFP: 8.5μl
T4 DNA ligase buffer: 2μl
T4 DNA ligase: 1μl
MilliQ water: 0.7μl

pSB1A3 self ligation control
pSB1A3-DT: 8.5μl
T4 DNA ligase buffer: 2μl
T4 DNA ligase: 1μl
MilliQ water: 8.5μl

pSB1A3+RBS-GFP
pSB1A3+RBS-GFP: 8.5μl
T4 DNA ligase buffer: 2μl
T4 DNA ligase: 1μl
MilliQ water: 8.5μl

Incubate for 30 minutes at roomtemperature.
Transformation:
- Add to 40μl competent cells 10μl DNA ligation mixture
- Incubate 30 minutes on ice
- Do the heatshock: incubate 1 min at 42°C
- Place the tubes on ice for 2 minutes and then pipette 1 ml of LB medium + 25mM glucose
- Incubate the tubes for 1 hour (with maximum of 1.5h) at 37°C
- Pellet the cells and resuspend them in 100μl medium
- Plate 90μl and 10μl of the cells on LB+ampicillin plates
- Incubate overnight at 37°C


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