Team:Heidelberg/Template/Del week18 Gibson

From 2013.igem.org

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:

• 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.