Team:Groningen/Notebook/10 July 2009
From 2009.igem.org
Glycerol Stocks
From the o.n. cultures of E.coli TOP10 with plasmids BBa_J23109, BBa_J23100 and BBa_J23106 glycerol stocks were made by adding 250μL of 87% sterile glycerol to 750μL of culture. The cells were frozen in liquid nitrogen and stored at -80°C.
Plasmid Purification
Plasmid isolation was performed on the cultures of promotor containing plasmids in cells with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".
- From each tube 2mL of culture was collected in a 2mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Cells were resuspended in 200μL Resuspension Solution by up and down pipetting.
- To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
- 350μL of Neutralisation Solution was added and the tubes inverted.
- Cell debri was pelleted by centrifugation at full speed for 1 min.
Concentration of Plasmids
The concentration of isolated plasmid was determined with the use of a nano-drop.
BBa_J23109 eluted in MQ
- 61.4 ng/μL
- 1.89 (260/280)
- 2.23 (260/230)
BBa_J23100 eluted in MQ
- 96.1 ng/μL
- 1.88 (260/280)
- 2.22 (260/230)
BBa_J23106 eluted in MQ
- 55.4 ng/μL
- 1.84 (260/280)
- 2.22 (260/230)
Restriction analysis
The plasmids containing GVP were cut with EcoRI and XbaI fast digest enzymes. The double digestion should result in two fragments of 1400 and 8000 bp in size.
- 6μL MQ
- 10μL plasmid in MQ
- 2μL Fast digest buffer
- 1μL EcoRI fast digest enzyme
- 1μL XbaI fast digest enzyme
The plasmids containing BBa_J23109, BBa_J23100, and BBa_J23106 were cut with EcoRI and PstI fast digest enzymes. The double digestion should result in two fragments of 1400 and 8000 bp in size.
- 0μL (8μL) MQ
- 16μL (8μL) plasmid in MQ
- 2μL Fast digest buffer
- 1μL EcoRI fast digest enzyme
- 1μL PstI fast digest enzyme
The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforese.
Gel electroforese
10μL of each sample (25μL for GVP) was loaded on a 2% agarose gel with EtBr and a 1kb ladder was used (see picture).
Gel Purification of GVP
A standard kit for PCR-product purification was used for gel purification
- ..mg of gel containing the desired fragment was dissolved in ..μL binding buffer by heating to ..°C for ..min.
- the column was prepared with ..μL
PCR HmtA Mut1/Mut2
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|