Team:Exeter/lab book/proto/13

Revision as of 14:56, 26 September 2012 by Agb207 (Talk | contribs)

Protocol 13


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase

SDS-PAGE Protocol
SDS-PAGE was run using 10% NuPAGE^® Novel^® Bis-Tris Mini Gels. Prepare a master-mix containing (procedure for 1 sample only): 2x NuPAGE^® LDS Sample Buffer (4x provided) 2x Sample Reducing Buffer (10x provided) Top up to 50µL using MilliQ H₂O Heat samples at 70^oC for 10 minutes. Prepare 1 x SDS Running Buffer by adding 50mL 20 x NuPAGE^® MES or MOPS SDS Running Buffer to 950mL of deionised water. Load the appropriate concentration of your protein sample on the gel. To load the buffer, fill the Upper (inner) Buffer Chamber with 200mL 1 x NuPAGE^® SDS Running Buffer. Fill the Lower (outer) Buffer Chamber with 600mL 1 x NuPAGE^® SDS Running Buffer. Then run the SDS-PAGE gel at 200V constant for 35 minutes (for MES Buffer) or 50 minutes (for MOPS Buffer). Once finished, stain using SimplyBlue^™ SafeStain, leave for approximately an hour, and then wash using MilliQ H₂O for direct visualisation.