Team:Warsaw/Calendar-Main/28 September 2009
From 2009.igem.org
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+ | ===<div style="text-align: center;"><h3>Bistable switch testing</h3></div>=== | ||
+ | |||
+ | '''<h4>Monika</h4>''' | ||
+ | |||
+ | Task: Control bistable switch | ||
+ | |||
+ | Methods | ||
+ | * Reaction mixture composition: | ||
+ | 6μl plasmid solution | ||
+ | 0,3μl HindIII (Fermentas) | ||
+ | 0,3μl NheI (Fermentas) | ||
+ | 2μl Buffer Tango (Fermentas) | ||
+ | 11,4 μl MQ water | ||
+ | |||
+ | * Digestion in 37°C for 2 h | ||
+ | |||
+ | {| class="wikitable" style="text-align:center; width:500px; height:90px; border-collapse: collapse; border: 1px dashed blue; margin: 1em auto 1em auto" border="1" | ||
+ | |- | ||
+ | ! DNA sample | ||
+ | ! restriction enzymes | ||
+ | ! expected fragments [bp] | ||
+ | |- | ||
+ | | Bistable switch | ||
+ | | HindIII, NheI | ||
+ | | 2700, 2200, 1800, 375, 200 | ||
+ | |} | ||
+ | |||
+ | Results | ||
+ | * Will be seen tomorrow | ||
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{{WarNotebookEnd}} | {{WarNotebookEnd}} |
Revision as of 19:01, 30 September 2009
Contents |
Assembly of endosome detection operon
Marcin
Task 1: Prepare the following ligations:
- [http://partsregistry.org/Part:BBa_K177035BBa_K177035] with [http://partsregistry.org/Part:BBa_K177036BBa_K177036] to obtain [http://partsregistry.org/Part:BBa_K177037BBa_K177037]
- [http://partsregistry.org/Part:BBa_K177035BBa_K177035] with [http://partsregistry.org/Part:BBa_K177043BBa_K177043] to obtain [http://partsregistry.org/Part:BBa_K177044BBa_K177044]
Methods:
- Reaction mixtures composition:
45 μl mixture of insert and vector DNA (both are purified at the same probe) 1.5 μl T4 ligase (Fermentas) 5 μl Ligase Buffer (Fermentas)
- Ligation was carried out 6 hours in 16 °C. Subsequently both mixtures were divided up two portions. In the case of each reaction one portion was thermally inactivated and the latter one was unremoved.
Task 2: Transformation of TOP10 chemocompetent bacteria with following constructs:
- [http://partsregistry.org/Part:BBa_K177044BBa_K177044]
- [http://partsregistry.org/Part:BBa_K177037BBa_K177037]
Methods:
- Ligation mixture was thermally inactivated
- Detailed protocol of transformation is described here
PCR of phoP and phoQ
Monika
Task:
- amplification of phoP (675 bp) and phoQ (1464 bp)
Methods:
- PCR mixture:
1μl Pfu polymerase buffer 0,2μl forward primer and 1μl reverse primer 0,4μl dNTPs (10 mM) 0,5μl Pfu turbo polymerase (EURX) 0,4μl template DNA from Salmonella enterica typhimurium LT2 The solution was topped up with H2O to 10μl contol - no template DNA from Salmonella enterica typhimurium LT2
- PCR conditions:
1. 3min 95°C 2. 30s 95°C 3. 35s 58°C 4. 2min 10 s 72°C 5. go to step 2, 2 times 6. 30s 95°C 7. 30s 68°C 8. 2min 10s 72°C 9. go to step 6, 28 times 10. 10min 72°C 11. forever 4°C
Results of PCR:
Bistable switch testing
Bistable switch testing
Monika
Task: Control bistable switch
Methods
- Reaction mixture composition:
6μl plasmid solution 0,3μl HindIII (Fermentas) 0,3μl NheI (Fermentas) 2μl Buffer Tango (Fermentas) 11,4 μl MQ water
- Digestion in 37°C for 2 h
DNA sample | restriction enzymes | expected fragments [bp] |
---|---|---|
Bistable switch | HindIII, NheI | 2700, 2200, 1800, 375, 200 |
Results
- Will be seen tomorrow
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