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Team:Warsaw/Calendar-Main/8 September 2009
From 2009.igem.org
(Difference between revisions)
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| + | <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> | ||
| + | <h4>Marcin</h4> | ||
| + | Task 1: Isolation of following construct from liquid bacterial culture: | ||
| + | * [http://partsregistry.org/Part:BBa_B0032<span style="color: black">BBa_B0032</span>] with [http://partsregistry.org/Part:BBa_E0022<span style="color: black">BBa_E0022</span>] connected to [http://partsregistry.org/Part:BBa_B0032<span style="color: black">BBa_B0032</span>] with [http://partsregistry.org/Part:BBa_C0051<span style="color: black">BBa_C0051</span>] on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] plasmid | ||
| + | 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: | ||
| + | <pre> | ||
| + | 2 μl purified plasmid DNA product | ||
| + | 1 μl PstI (Fermentas) | ||
| + | 1 μl XbaI (Fermentas) | ||
| + | 2 μl Buffer Tango (Fermentas) | ||
| + | 15 μl MQ water | ||
| + | </pre> | ||
| + | * Digest was performed 3 hours | ||
| + | Results: | ||
| + | |||
| + | Task 3: | ||
| + | * Digest of folowing constructs ( both of them are on [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] plasmid): | ||
| + | **[http://partsregistry.org/Part:BBa_B0032<span style="color: black">BBa_B0032</span>] with [http://partsregistry.org/Part:BBa_E0022<span style="color: black">BBa_E0022</span>] | ||
| + | **[http://partsregistry.org/Part:BBa_B0032<span style="color: black">BBa_B0032</span>] with [http://partsregistry.org/Part:BBa_C0051<span style="color: black">BBa_C00251</span>] | ||
| + | Methods: | ||
| + | * Reaction mixture composition: | ||
| + | <pre> | ||
| + | 1. μl purified plasmid DNA product | ||
| + | 1 μl XbaI (Fermentas) | ||
| + | 1 μl PstI (Fermentas) | ||
| + | 2 μl Buffer Tango (Fermentas) | ||
| + | 15 μl MQ water | ||
| + | </pre> | ||
| + | * 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. | ||
| + | |||
| + | |||
| + | <h3><div style="text-align: center;">Assembly of fusion proteins</div></h3> | ||
| + | <h4>Marcin</h4> | ||
| + | |||
| + | 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 [https://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009 here] | ||
Revision as of 08:01, 9 September 2009
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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
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