SDS Page-Gel for all the bovine casein inserts

17 January 2015: SDS Page-Gel All 5 Bovine Casein Inserts

Location: BioCurious

Participants: Lafia, Johan, Meenakshi, Emi, Rachel

Notes: Emi

Aims: Confirmation of bovine casein inserts from liquid culture of yeast transformed cells

Materials

Gel loading tips

Tris-Acelate SDS Running Buffer novex life tech

Bio-Rad pipette tips

Native gels... (need details)

ladder 20x

Casein

Cytosin

Transformed negative control

Bov alpha s1 (1) Transformed liqid culture

Bov beta B (8) Transformed liquid culture

Bov alpha S2 (3) Transformed liquid culture

Bov beta A2 (6B) Transformed liquid culture

Bov kappa (7F) Transformed liquid culture

Chymosin + casein (Chymosin cuts casein and separates it to 2 bands)

Ladder

Protocol

Centrifuge the falcon tubes, 5 minutes 2800xg

Take 500ul of distilled water in eppendorf tube + 1 tiny drop of casein (use 200ul hp take it) - 2times

Take 15ul of chymosin +1 tiny drop of casein in 250ul of water

Take 250ul of 7F (Bov Kappa) + 15ul of Chymosinin Take 250ul of water and 15ul of chymosin

Take 40ul of 1, 8, 3B, 6B, +7F also the negative control to eppendorf tubes

Preparation fo 20x running buffer

Pour 50ul of 10x running buffer; make it to 450ul by adding the distilled water - total 500ul

20ul of loading dye to each tubes except casein; chymosin and casein; chymosin and 7F

Thus we have to add total 120ul of dye

Boil all the tubes for 10 mins at 95C (inorder to denature the protein structure to linear) (only