Team:Heidelberg/Template/Del week18 Gibson

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==30-08-2013==
==30-08-2013==
===SDS-PAGE===
===SDS-PAGE===

Latest revision as of 12:44, 3 October 2013

Contents

30-08-2013

SDS-PAGE

Lysis of cells

  • 1 ml over night cultures in LB of electrocompetent DH10ß, DH10ß with pSB4K5 and DH10ß with pFSN
  • meausured ODs (DH10ß= 1,32; DH10ß+pSB4K5= 1,28; DH10ß+pFSN= 1,12)
  • centrifugation at 13000 rpm for 10 min
  • discarded supernatant
  • resuspended in 66 µl(DH10ß), 64 µl(DH10ß+pSB4K5) and DH10ß+pFSN= 56 µl 1x loading buffer (40% ddH2O, 12.5% TrisHCl pH=7.5, 20% Glycerol (50%), 20% SDS (10%), 5% β-Mercaptoethanol, 2.5% Bromophenol blue)
  • boiled samples for 10 min at 98°C

Electrophoresis

  • Applied 2.5 µl and 10 µl of each sample on SDS-polyacrylamide gel
  • Let it run for 1 h at 180 V

Staining and destaining procedure

  • Gel was stained in Coomassie Brilliant Blue for 1 h on shaker
  • Destaining with destaining buffer (50% ddH2O, 40% Methanol, 10% Acetic acid) until bands were visible

Results:

SDS-PAGE of the samples DH10β, DH10β+pSB4K5 and DH10β+pFSN; arrow indicates band of DelE
  • One band at a height of about 190 kDa was visible in DH10ß+pFSN, but not in the controls. As the protein DelE has a molecular weight of 192.52 kDa this band is probably DelE.
  • A very weak band could be detected well above the 260 kDa band of the ladder. We suspect that this might be DelG, as it has a molecular weight of 357.33 kDa, however the band was too weak to capture it in a picture. Thus a bigger amount of the samples have to be applied on a SDS-PAGE again.
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