Team:Warsaw/Calendar-Main/3 September 2009
From 2009.igem.org
Contents |
Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek
Tasks:
- Digestion of PCR products, separetly with MboI and CaiI restriction endonucleases
- Electrophoretical segregation of digested samples in 1,5% agarose gel
- Preparation of another PCR reaction with L.monocytogenes genome DNA
Results:
- There were no visible products of digestion, even after long treatment with ethidium bromide. Propably the Clean-Up kit isn't working well. I'll try to acquire IntA gene with another PCR.
Preparation of glycerol stocks
Preparation of glycerol stocks
Monika
- Preparation of glycerol stocks following the prodcedure described here
pAraC + GFP
plac + RBS + cI + ter
plac + RBS + cI + RBS + GFP + ter
J07037
plac
pSB6A1
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmids containing following construct:
- BBa_K177035 on pSB1A3 plasmid
Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Task 2:
- Digestion of plasmids previously described constructs
Methods:
- Reaction mixture composition:
20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
- Digest was performed about six hours and subsequently thermal inactivated
Task 3:
- Gel-outs of construct described in Task 2
Methods:
- All gel-outs were performed using the EurX gel-out kit according to the manual
Task 4:
- Ligation of following constructs:
- BBa_K177037 on pSB1A3 plasmid
- cro CDS on pSB1A3
- Bax CDS on pSB1A3
Methods:
- Reaction mixture composition:
10 μl digested insert 8 μl digested vector 2 μl Tango buffer(Fermentas) 3 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas)
- Reaction were carried out 12 hours and subsequently thermally inactivated.
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Plasmid isolation
- Control digest
Methods:
- Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
- The digest mix was prepared as follows:
20μl isolated plasmid
3μl Buffer O (Fermentas)
1μl PstI enzyme
1μl EcoRI enzyme
5μl H2O - The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
- The results were visualised on 1% agarose gel
Results:
- First four bands (besides the the size ruler) show empty plasmids.
Conclusions:
- Respawn and try again in... 3 days.
Cloning of the cro-box into the pSB1A3 plasmid
Kamil
Tasks:
- Plasmid isolation
- Control digest
Methods:
- Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).
- The digest mix was prepared as follows:
20μl isolated plasmid
3μl Buffer O (Fermentas)
1μl PstI enzyme
1μl EcoRI enzyme
5μl H2O - The digest was carried out for 3h in 37°C and then inactivated in 80°C for 15min.
- The results were visualised on 1% agarose gel
Results:
- Final five bands show empty plasmids.
Conclusions:
- Respawn and try again in... 3 days.
Cotransformation to obtain cells with following plasmid combinations [K177012,K177011][K177012,K177038][K177033,K177011][K177033,K177038]
Ania
Tasks:
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Ania
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