IGEM:Groningen/Notebook/iGEM 2011/2011/07/18

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18-7-11


Today, Digestion, DNA clean up with High Pure PCR purification kit of Roche, ligation, transformation to create transformants
with: PBAD-LasR-LVA-DT and PhybB-LasR-LVA-DT
Also, colony of PBAD-RBS-GFP and PBAD-cI-LVA-DT were grown overnight in LB medium with ampicillin and this morning, the culture had been grown, so plasmid prep, glycerol stock and sample for sequencing should be made.
Digestion:
LasR-LVA-DT vector
2μl vector
1μl EcoR1
1μl XbaI
3μl Fast digest buffer
1μl Fast AP
22μl 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-LasR-LVA-DT vector
8.5μl vector
6.1μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.4μl MQ

PhybB-LasR-LVA-DT vector
8.5μl vector
4.1μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
4.4μl 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)

Colony PCR with samples PBAD-cI-LVA-DT, PhybB-cI-LVA-DT and PBAD-RBS-GFP-DT (samples ligation mixtures stored overnight in fridge)was done as followed:
Mastermix scheme:
Taq 10× buffer with NH4SO4 without MgCl2: 58.00μl
dNTPs 10mM each: 11.6μl
MgCl2: 34.8μl
Taq polymerase 5u/μl: 2.90μl
Biobrick vector forward primer 10μM: 11.6μl
Biobrick vector reverse primer 10μM: 11.6μl
MilliQ water: 449.50μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle (33×)
Denaturation: 94°C for 30s.
Annealing: 60°C for 30s.
Extension: 72°C for 2 min.
Final extension: 72°C for 10 min.
Store at 4°C infinite

PCR products were analysed on a 1% agarose gel with TBE.
Results: PBAD-cI-LVA-DT colony 1 seems to be right (correct size on gel (2kb) )
PhybB-cI-LVA-DT can be right, is difficult to see, maybe use one colony and tomorrow (with other colony PCR probably, pick
some of the old plates of this sample aswell.
PBAD-RBS-GFP-DT colony 4 looks alright, but is difficult to see because the gel is not very good, so just try along with the
pBAD-cI-LVA no right transformants, but the one of friday will be used as the sample send for sequencing.

PCR for PhybB PCR product (there is not much left anymore...)

PCR was done as followed:
Mastermix scheme:
Taq 10× buffer with NH4SO4 without MgCl2: 25.00μl
dNTPs 10mM each: 5μl
MgCl2: 15μl
Taq polymerase 5u/μl: 1.25μl
Pfu polymerase: 0.2μl
Forward primer PhybB: 5μl
Reverse primer PhybB: 5μl
MilliQ water: 193.55μl
End volume in PCR tube: 50μl.

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle (37×)
Denaturation: 94°C for 30s.
Annealing: 65°C and 70°C (gradient) for 30s.
Extension: 72°C for 2 min.
Final extension: 72°C for 10 min.
Store at 4°C infinite (overnight)

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