Team:Groningen/Notebook/10 July 2009

From 2009.igem.org

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'''Gel Purification of GVP'''
'''Gel Purification of GVP'''
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A standard kit for PCR-product purification was used for gel purification...
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A standard kit for PCR-product purification was used for gel purification  
 +
 
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* ..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'''
'''PCR HmtA Mut1/Mut2'''

Revision as of 13:02, 10 July 2009

Igemhomelogo.png

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



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November
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