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Team:Warsaw/Calendar-Main/14 September 2009
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(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3>Cloning of cro gene into pSB+ vector</h3> <h4>Kama</h4> <ul> <li>chemocompetent E. coli TOP10 were incubated on ice for 15 minutes...) |
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| + | <h3>Assembly of endosomal detection operone/Assembly of fusion proteins</h3> | ||
| + | <h4>Marcin</h4> | ||
| + | Task 1: | ||
| + | * Digest plasmids isolated in [https://2009.igem.org/Team:Warsaw/Calendar-Main/13_September_2009 13.09.2009] to reveal the identity of the constructs | ||
| + | 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> | ||
| + | * Digestion was carry out 2 hours | ||
| + | Results: | ||
| + | There is no evidence for occurence of signal peptide CDS. The situation of [http://partsregistry.org/Part:BBa_K177037<span style="color: black">BBa_K177037</span>] biobricks is unclear since lack of expected restriction pattern | ||
| + | '''Comment:''' | ||
| + | Because of short length of signal peptide it is possible that I am incapable of finding this fragment on the gel. I think the sequencing of some samples may reveal the identity of the both signal peptide and [http://partsregistry.org/Part:BBa_K177037<span style="color: black">BBa_K177037</span>] | ||
| + | Task 2: | ||
| + | |||
| + | '''Comment:''' | ||
| + | |||
| + | Due to some problem which our ampicilline I decided to use vector with resistance to kanamycine instead of previously use [http://partsregistry.org/Part:pSB1A3<span style="color: black">pSB1A3</span>] | ||
| + | |||
| + | * Prepare the backbone plasmid [http://partsregistry.org/Part:pSB1K3<span style="color: black">pSB1K3</span>] to ligation with COX mitochondrial signal. | ||
| + | |||
| + | * Reaction mixture composition: | ||
| + | <pre> | ||
| + | 20 μl purified plasmid DNA product | ||
| + | 1 μl PstI (Fermentas) | ||
| + | 1 μl SpeI (Fermentas) | ||
| + | 5 μl Buffer Tango (Fermentas) | ||
| + | 23 μl MQ water | ||
| + | </pre> | ||
| + | * Digestion was carry out 4 hours | ||
| + | |||
| + | Result: | ||
| + | |||
| + | The digestion was severe incomplete. Most of DNA was uncut. It is obligated to carry out the reaction longer. | ||
Latest revision as of 11:25, 18 September 2009
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Cloning of cro gene into pSB+ vector
Kama
- chemocompetent E. coli TOP10 were incubated on ice for 15 minutes
- 20μl of ligation mixture was added
- bacteria were incubated with DNA on ice for 30 minutes
- heat shock was conducted (1min30sec 42°C)
- bacteria were incubated on ice for 3 minutes
- After the heat shock 780μl of SOB medium was added
- Mixture with bacteria was incubated for 1 hour in 37°C
- 100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG (diluted 10* and without dilution)
Assembly of endosomal detection operone/Assembly of fusion proteins
Marcin
Task 1:
- Digest plasmids isolated in 13.09.2009 to reveal the identity of the constructs
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
- Digestion was carry out 2 hours
Results: There is no evidence for occurence of signal peptide CDS. The situation of [http://partsregistry.org/Part:BBa_K177037BBa_K177037] biobricks is unclear since lack of expected restriction pattern
Comment:
Because of short length of signal peptide it is possible that I am incapable of finding this fragment on the gel. I think the sequencing of some samples may reveal the identity of the both signal peptide and [http://partsregistry.org/Part:BBa_K177037BBa_K177037]
Task 2:
Comment:
Due to some problem which our ampicilline I decided to use vector with resistance to kanamycine instead of previously use [http://partsregistry.org/Part:pSB1A3pSB1A3]
- Prepare the backbone plasmid [http://partsregistry.org/Part:pSB1K3pSB1K3] to ligation with COX mitochondrial signal.
- Reaction mixture composition:
20 μl purified plasmid DNA product 1 μl PstI (Fermentas) 1 μl SpeI (Fermentas) 5 μl Buffer Tango (Fermentas) 23 μl MQ water
- Digestion was carry out 4 hours
Result:
The digestion was severe incomplete. Most of DNA was uncut. It is obligated to carry out the reaction longer.
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