1 / 11

Western Analysis/RNA isolation

Western Analysis/RNA isolation. Mod2 Day5 Tuesday Oct 21st. About antibodies. Polyclonal Monoclonal. The 1° antibody you’ll use…. The 2° antibody you’ll use…. Yfgp. Part 1: Western Analysis. If Yfg = 25 kD + TAP tag = ~20 kD. Yfgp. Part 1: Western Analysis--what if?. If Yfg = 25 kD

donaldg
Download Presentation

Western Analysis/RNA isolation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related