IGEM:Groningen/Notebook/iGEM 2011/2011/07/19

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overnight of PCR of PhybB worked-> purified with roche kit and concentration is about 55 ng/μl according to the nanodrop system.
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 PCR of PhybB-cI-LVA-DT
Digestion of insert needs to be done with non fast digest enzymes, since SpeI is not present as fast digest enzyme, so SpeI is
used with Tango buffer and so EcoRI also needs to be used in this case as a non fast digest enzyme.
LasR-LVA-DT vector
2μl vector
1μl EcoR1
1μl XbaI
3μl Fast digest buffer
1μl Fast AP
22μl MQ

10μl pBAD
1μl SpeI
2μl Tango buffer
7μl MQ

3μl PhybB
1μl SpeI
2μl Tango buffer
14μl MQ
Incubate the samples at 37°C for 1.5h.
After this, you have to add EcoRI (1μl) and 20/8=2.5 μl Tango buffer.
Incubate another time at 37°C for 1.5h. For more information see: http://www.fermentas.com/en/tools/doubledigest
Do a DNA purification with the PCR purification kit from Roche

PhybB-LasR-LVA-DT vector
8.5μl vector
5.6μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.9μl MQ

PBAD-LasR-LVA-DT vector
8.5μl vector
6.1μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.4μl MQ

PhybB-cI-LVA-DT vector
8.5μl vector
5.6μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.9μl 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)

Colony PCR with samples PhybB-cI-LVA-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.
PhybB-cI-LVA-DT can be right, is difficult to see, maybe use some colonies-> 4 were used for overnight culture so tomorrow a
plasmid prep should be done.