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. Part 1: Western Analysis
Yfgp
If Yfg = 25 kD
+ TAP tag = ~20 kD
Dalton = an atomic mass unit = mass of 1 H atom

6. Part 1: Western Analysis--what if?
Yfgp
If Yfg = 25 kD
+ TAP tag = ~20 kD
Dalton = an atomic mass unit = mass of 1 H atom

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
Lab will meet in 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