Team:Groningen/Notebook/10 July 2009

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Revision as of 14:13, 10 July 2009

Glycerol Stocks

From the o.n. cultures of E.coli TOP10 with plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 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.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] eluted in MQ

• 61.4 ng/μL
• 1.89 (260/280)
• 2.23 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] eluted in MQ

• 96.1 ng/μL
• 1.88 (260/280)
• 2.22 (260/230)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100], and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 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).

→ The fragments of GVP were of the expected size for a double cut, for all three promotor containing plasmids three bands were seen on the gel. This would correspond to a single cut upper band of about 3000bp and two bands for the double cut parts of 1000bp and 2000bp. Melbourne already mentioned trouble with digestion with PstI, and could be the problem here. For the assembly into the GVP-plasmid, restriction with SpeI, instead of PstI, is required and might be better in completely cutting the plasmid

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