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Team:Warsaw/Calendar-Main/20 July 2009
From 2009.igem.org
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Insertion of the pho gene into the pKSII+ plasmid
Kama
- Plasmid digest mix was prepared as follows:
3μl Tango buffer (Fermentas) 4μl pKSII plasmid 1μl XbaI enzyme 1μl SmaI enzyme
The solution was topped up with H2O to the final volume of 30 μl. - Pho gene digest mix was prepared as follows:
3μl Tango buffer (Fermentas) 16μl purified gene 1μl XbaI enzyme
The solution was topped up with H2O to the final volume of 30 μl. - The digest was kept for 2h at 30°C, after that 2h at 37°C and then the enzymes were inactivated for 20min. at 70°C.
The ligation mix was prepared as follows:
2μl plasmid 8μl gene 3μl ligation buffer 1 μl T4 DNA ligase (Fermentas) 0,5μl PEG
The solution was topped up with H2O to the final volume of 30 μl. As a control:2μl plasmid 3μl ligation buffer 1 μl T4 DNA ligase (Fermentas) 0,5μl PEG
The solution was topped up with H2O to the final volume of 30 μl. The ligation was carried out in 18°C overnight (~15h)
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Set up a liquid culture from transformants containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid in order to amplify the plasmid and extract it from the bacteria.
Comment:
- Accidentally I used the whole DNA samples extracted on Thursday to perform the digestion. I Added the digestion mix to my samples, hence the wrong buffer concentration and improper digestion on Friday. Thinking saves time and money. But there is another reason to justify the repeated plasmid extraction. Now I know that my transformants after all have the correct plasmid so I can do the alkaline lysis on very few samples and use the AA kit to get greater DNA concentration. After all this is my insert for the next ligation, so we need a lot. I know that no one will ever read the notebook up to this point.
Testing different E. coli strains regarding [http://partsregistry.org/Part:BBa_R0010 lacI] and [http://partsregistry.org/Part:BBa_R0080 AraC] repressors
Franek
Tasks:
- Seting liquid cultures of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Methods:
- 7 test tubes with 5ml LB were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a. The cultures were left for overnight incubation at 37°C.
- 14 test tubes with 5ml LB and ampicillin were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing either [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] or [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C.
- 7 test tubes with 5ml LB, ampicillin and IPTG were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C.
- 7 test tubes with 5ml LB, ampicillin and 0,2% arabinose were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C.
Results:
- Will be determined next day by comparing GFP level in diffrent cultures
Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Plasmid assembly
Methods:
- Plasmid digest mix was prepared as follows:
2μl Tango buffer (Fermentas) 3μl pKSII+ plasmid 2μl XbaI enzyme (Fermentas) 2μl SmaI enzyme (Fermentas)
The solution was topped up with H2O to the final volume of 20 ul. - mgtc promoter digest mix was prepared as follows: 2μl Tango buffer (Fermentas) 10μl purified gene 2μl XbaI enzyme The solution was topped up with H2O to the final volume of 20 ul.
- The digest was kept for 6h (to compensate for the 50% loss in activity of the SmaI enzyme) at 37°C, and then the enzyme was inactivated for 15min. at 80°C.
The ligation mix was prepared as follows:
10μl of both inactivated digests were mixed together and topped with 2μl of 30% PEG 2,2μl ligation buffer (containing ATP) 2μl of T4 ligase (Fermentas)
. The ligation was carried out in 18°C overnight (~18h) and then inactivated for 10min. at 65°C.
Results:
From left:
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- Ligation
- Undigested plasmid
Conclusions:
- Despite the low morale we decided we could transform with that.
Assembly of endosomal detection operon
Marcin
Task 1:
- Inactivation of ligation
Ligation mixtures prepared 19.07.09 were inactivation via heating in 80°C for 20 minutes.
Comment:
Due to some doubts about quality of the chemocompetent bacteria I decided to freeze the ligation mixtures until the situation would become more clear.
Cloning of p53 coding sequence
Marcin
Task 1:
- Prepare PCR reaction to amplified p53 coding sequence.
Methods:
- DNA template dilution:
1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water
- PCR mixture composition:
- proper mixture 1:
0.25 μl primer 1 (50 nM; Oligo.pl) 0.25 μl primer 2 (50 nM; Oligo.pl) 1.5 μl dNTPs (20 μM ;Fermentas) 0.5 μl Pfu turbo polymerase (KNGiE) 2.5 μl Pfu Turbo Buffer (Fermentas) 2.5 μl MgSO4 (20 μM; Fermentas) 1 μl DNA template 16.5 μl MQ water
- proprer mixture 2:
0.25 μl primer 1 (50 nM; Oligo.pl) 0.25 μl primer 2 (50 nM; Oligo.pl) 1.5 μl dNTPs (20 μM ;Fermentas) 0.5 μl Pfu turbo polymerase (KNGiE) 2.5 μl Pfu Turbo Buffer (Fermentas) 2.5 μl MgSO4 (20 μM; Fermentas) 2 μl DNA template 15.5 μl MQ water
- Negative control: the same as proper mixture 1, the only distinction is lack of the DNA template.
- Program:
p53 (detailed destription is here)
Results:
No product obtained
Comment:
Previous PCR reaction were prepared using another thermocycler with distinct intristic parameter than used in this case. Perhaps, if the reactions were prepared in that device the results would be better.
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