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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.
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Methods:- <p>PCR mixture's composition:</p> 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. <p>One of the samples also contained 1 ul of DMSO</p>
- PCR programs:
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
- Electrophoretic separation on 1% agarose gel
- 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)
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
no siginificant amounts of desired product were obtained
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