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Team:Warsaw/Calendar-Main/7 July 2009
From 2009.igem.org
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Yet another insertion of the mgtc gene into the pKSII+ plasmid
Kamil
Tasks:
- Plasmid assembly
Methods:
The ligation mix was prepared as follows: 1ul plasmid, 3ul gene, 1ul ligation buffer B (Fermentas), 1ul T4 DNA ligase (Fermentas), 1ul 30% PEG, 1ul 10mM ATP, the solution was topped up with H2O to the final volume of 10 ul. The ligation was carried out in 37°C for 1h and then inactivated for 15min. at 65°C.
A 200ul batch of chemocompetent bacteria was transformed with 3ul of ligation mix and incubated on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.
PCR mixture's composition:
2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul.One of the samples also contained 1 ul of DMSO
- PCR programs:
UPDATE: No luck this time, back to the drawing board...
Yet another attempt at obtaining a specyific Llo PCR product for fusion with the secretion signal
Kuba
PCR was preformed on a product of classical biobrick-format Llo (reamplification)
three reactions were preformed, each with increasing concentration of template
Methods:
hly
4min 95°C
(30s 95°C, 40s 41°C, 1min40s 72°C)x3
(30s 95°C, 40s 44°C, 1min40s 72°C)x28
10min 72°C
~ 7°C
random program
- Electrophoretic separation on 1% agarose gel
Results:
- Gel (from left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- sample no.1 (1ul of 100-fold diluted template)
- sample no. 2 (2ul of 100-fold diluted template)
- sample no. 3 (1ul of template)
no siginificant amounts of desired product were obtained
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