IGEM:Groningen/Notebook/iGEM 2011/2011/07/19: Difference between revisions

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==Entry title==
==19-7-11==
* Insert content here...
<br> overnight of PCR of PhybB worked-> purified with roche kit and concentration is about 55 ng/μl according to the nanodrop system.
 
<br> Digestion, DNA clean up with High Pure PCR purification kit of Roche, ligation, transformation to create transformants <br> <br> with: PBAD-LasR-LVA-DT and PhybB-LasR-LVA-DT
 
<br> Also, colony PCR of PhybB-cI-LVA-DT
<br> Digestion:
<br> Digestion of insert needs to be done with non fast digest enzymes, since SpeI is not present as fast digest enzyme, so SpeI is <br> used with Tango buffer and so EcoRI also needs to be used in this case as a non fast digest enzyme.
<br> LasR-LVA-DT vector
<br> 2μl vector
<br> 1μl EcoR1
<br> 1μl XbaI
<br> 3μl Fast digest buffer
<br> 1μl Fast AP
<br> 22μl MQ
<br>
<br> pBAD
<br> 10μl pBAD
<br> 1μl SpeI
<br> 2μl Tango buffer
<br> 7μl MQ
<br>
<br> PhybB
<br> 3μl PhybB
<br> 1μl SpeI
<br> 2μl Tango buffer
<br> 14μl MQ
<br> Incubate the samples at 37°C for 1.5h.
<br> After this, you have to add EcoRI (1μl) and 20/8=2.5 μl Tango buffer.
<br> Incubate another time at 37°C for 1.5h. For more information see: http://www.fermentas.com/en/tools/doubledigest
<br> Do a DNA purification with the PCR purification kit from Roche
<br> Ligation:
<br>
<br> PhybB-LasR-LVA-DT vector
<br> 8.5μl vector
<br> 5.6μl insert
<br> 1μl T4 DNA ligase
<br> 2μl T4 DNA ligase buffer
<br> 2.9μl MQ
<br>
<br> PBAD-LasR-LVA-DT vector
<br> 8.5μl vector
<br> 6.1μl insert
<br> 1μl T4 DNA ligase
<br> 2μl T4 DNA ligase buffer
<br> 2.4μl MQ
<br>
<br> PhybB-cI-LVA-DT vector
<br> 8.5μl vector
<br> 5.6μl insert
<br> 1μl T4 DNA ligase
<br> 2μl T4 DNA ligase buffer
<br> 2.9μl MQ
<br>
<br>Self ligation:
<br> 8.5μl vector
<br> 1μl T4 DNA ligase
<br> 2μl T4 DNA ligase buffer
<br> 8.5μl MQ
<br>
<br> Ligate at room temperature for 30 min.
<br>
<br>Transform as usual (see 12th of July)
<br>
<br>
<br> Colony PCR with samples PhybB-cI-LVA-DT (samples ligation mixtures stored overnight in fridge)was done as followed:
<br> Mastermix scheme:
<br> Taq 10× buffer with NH4SO4 without MgCl2: 58.00μl
<br> dNTPs 10mM each: 11.6μl
<br> MgCl2: 34.8μl
<br> Taq polymerase 5u/μl: 2.90μl
<br> Biobrick vector forward primer 10μM: 11.6μl
<br> Biobrick vector reverse primer 10μM: 11.6μl
<br> MilliQ water: 449.50μl
<br>
<br> PCR conditions:
<br> Preheated lid: 111°C
<br> Denaturation: 94°C for 10 min.
<br> Cycle (33×)
<br>  Denaturation: 94°C for 30s.
<br>  Annealing: 60°C for 30s.
<br>  Extension: 72°C for 2 min.
<br> Final extension: 72°C for 10 min.
<br> Store at 4°C infinite
<br>
<br> PCR products were analysed on a 1% agarose gel with TBE.
<br> PhybB-cI-LVA-DT can be right, is difficult to see, maybe use one colony and tomorrow (with other colony PCR probably, pick <br> some of the old plates of this sample aswell.
<br>
<br>
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Revision as of 08:35, 19 July 2011

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19-7-11


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:
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

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

PhybB
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
Ligation:

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 one colony and tomorrow (with other colony PCR probably, pick
some of the old plates of this sample aswell.

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