Label membrane with pencil soak in meoh 15min gently

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**Wear gloves** Cut top right corner off. Label membrane with pencil. Soak in MeOH 15min. Gently separate gel from 1 plate, trim excess & stacking gel. Cut top right corner of gel. Flip as placed in transfer buffer in the next step Soak PVDF, fiber filters, & gel in transfer buffer 15 min. Add transfer buffer & roll out any bubbles between each layer. Layer on black side of holder: fiber, filter with gel, membrane, filter, fiber Place in holder black to black and clear to red. Repeat for second gel. Fill reservoir with transfer buffer. Place reservoir on stir plate, start magnet spinning, add holder. Run 20-24H Hook up red to red & black to black. Run 10V .1Amp max 4 C o/n ~24 hours. Mark ladder on membrane. Transfer Buffer 100 ml 10X Running Buffer 100 ml methanol Up to 1L with H 2 O
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Melissa Landis a512665f03678af630457fa7d4e5aa0d64c104be.doc 4/29/19 Semi-dry Transfer Equilibrate gel 2X15 in Transfer buffer Wet PVDF (14X16cm2) in MeOH 15 min, Transfer buffer 15 min Wet extra thick blotting paper in Transfer buffer Stack: extra thick blotting paper, pvdf, gel, extra thick blotting membrane o Add each buffer to each layer o Roll each layer with glass tube Transfer with semidry apparatus: 25V, 20min, .7Amp max
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Melissa Landis a512665f03678af630457fa7d4e5aa0d64c104be.doc 4/29/19 SDS-PAGE Project: Resolving Gel (2) 8% 10% 12% 14% Stacking Gel (2) 5% ml ml ml ml ml 30% acrylamide/bis 26.5 33.5 39.5 46.5 30% acrylamide/bis 8.3 ddH20 47 40 34 27 ddH20 34 1.5M Tris, pH 8.8 25 25 25 25 0.5M Tris, pH 6.8 6.3 20%SDS 0.5 0.5 0.5 0.5 20%SDS 0.25 10% APS 1 1 1 1 APS 0.5 TOTAL 100 100 100 100 TOTAL 49.35 Add 50 l TEMED to resolving gel. Add 40 l TEMED to stacking gel. Prepare samples & load samples Boil 3 minutes (because SDS precipitates when frozen), spin flash spin. Run 40 l prestained benchmark (invitrogen) marker. Run 35mAmps per gel (70 for 2) for ~1H until protein is through stacking gel. Run 50mAmps per gel (100 for 2).
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