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Comprehensive Guide to RNA Isolation and Western Analysis Techniques

Learn about antibodies, RNA isolation steps, sample selection, and crucial reminders for successful experimentation.

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Comprehensive Guide to RNA Isolation and Western Analysis Techniques

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  1. Western Analysis/RNA isolation Mod2 Day5 Tuesday Oct 21st

  2. About antibodies • Polyclonal • Monoclonal

  3. The 1° antibody you’ll use…

  4. The 2° antibody you’ll use…

  5. Yfgp Part 1: Western Analysis If Yfg = 25 kD + TAP tag = ~20 kD

  6. Yfgp Part 1: Western Analysis--what if? If Yfg = 25 kD + TAP tag = ~20 kD

  7. Part 2: RNA Isolation • Which samples will you choose?

  8. Part 2: RNA Isolation • Which samples will you choose? • Steps: • Calculate volume for 1x10^8 cells 5 OD600 = 1x10^8 cells/ml • Lyse cells with glass beads/vortex/PCI • Load lysate on column + wash • Elute with warm elution buffer • Measure concentration of 1:100 dil’n RNAs 1 A260 = 40 μg/ml of RNA

  9. Part 2: RNA Isolation • Things to remember • RNA is sensitive to RNAses that are everywhere… • Phenol-CHCl3-I will burn you if it gets on your skin, eyes, clothes… • Do NOT mix up the samples you load on the column… • The quartz cuvettes are fragile/expensive and the only ones we have…

  10. Hidden Part 3 • Scan or photograph your petri dishes from your spot tests which were pulled after 4 days at given temp. • We will scan your Westerns and post the data to the talk page for today’s wiki

  11. FNT • Lecture is optional trip to Abcam in Kendall Square, meet 11AM 66-168 • Lab will meet in 16-336 for journal club M2D5 FNT (due Thursday if not presenting) • Figure of spot tests • Figure for Westerns • Calculation of volume = 2 ug RNA • Mid-term evaluations (anon) M2D6 FNT (if you’re presenting) 2+2 due Friday by 5PM

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