Team:Warsaw/Calendar-Main/8 September 2009
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Revision as of 01:20, 10 September 2009
Cloning of the mgtc promoter into the pSB1A3 plasmid
Kamil
Tasks:
- Colonies transfer
- Liquid cultures establishment
Methods:
- Appropriate colonies (all 5 of them) were transferred to a new plate containing ampicilin.
- Liquid cultures were established in 4 ml LB medium supplemented with ampicilin and incubated overnight with areation.
Cloning of the cro-box into the pSB1A3 plasmid
Kamil
Tasks:
- Colonies transfer
- Liquid cultures establishment
Methods:
- Appropriate colonies (all 7 of them) were transferred to a new plate containing ampicilin.
- Liquid cultures were established in 4 ml LB medium supplemented with ampicilin and incubated overnight with areation.
Contents |
Assembly of endosomal detection operon
Marcin
Task 1: Isolation of following construct from liquid bacterial culture:
Methods:
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Task 2:
- Digest previously isolated plasmid to verify the correctness of the ligation:
Methods:
- Reaction mixture composition:
2 μl purified plasmid DNA product 1 μl PstI (Fermentas) 1 μl XbaI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
- Digest was performed 3 hours
Results:
Task 3:
- Digest of folowing constructs ( both of them are on pSB1A3 plasmid):
- BBa_B0032 with BBa_E0022
- BBa_B0032 with BBa_C00251
Methods:
- Reaction mixture composition:
1. μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
- Digestions were carry out 5 hours and subsequently were inactivated via heating for 20 minutes
Comment: The length of digested sequence is unexpected. The correct sequence should have had 1500 bp. Althought in each case the length is approximately 950 bp. It is obligated to carry out sequencing reaction to reveal the identity of this DNA.
Assembly of fusion proteins
Marcin
Task:1
- Transformation of chemocompetent E. coli TOP10 strain
Construct to transform:
- signal peptid from Cox1 CDS on pKSII vector
Methods:
- Ligation mixture was thermally inactivated in 65 °C
- Detailed protocol of transformation is described here
Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek
Tasks:
- Phosphorylation of PCR product with T4 phage kinase.
- Digestion of pUC18 with HincII endonuclease and dephosphorylation of it's ends with CIAP phosphatase.
- Seting ligation of internaline A with pUC18.
Inverting PLacI regulated parts of the switch and assembling the switch on a single plasmid
Ania
Tasks:
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April
M T W T F S S
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May
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June
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July
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August
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September
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October
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Ania
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