2021

2021 kdorfman Mon, 07/26/2021 - 16:51

Class Schedule:

date day cells reagents
9/2 Th fixed HeLa H2B-GFP mcherry-alpha Tubulin 10 untreated, and 24 treated with VPA, nocodazole, trichostatin A, cytochalasin D, latrunculin
9/7 Tu
9/9 Th
9/14 Tu PS-Speck slides B&G
9/16 Th
9/21 Tu photo bleaching HeLa slides
9/23 Th B16 on coverslips Para-glut
Rat anti-tubulin
goat anti-rat
PBS-Tw-Az
9/28 Tu 1 flask B16/pair passaging
9/30 Th view dish from previous lab
split for nucleofection		 mini-prep EB1
10/5 Tu
10/7 Th
10/12 Tu
10/14 Th
10/19 Tu
10/21 Th
10/26 Tu Students split cultures from Drew into 4-chambered dishes
10/28 Th
11/2 Tu Students split onto 4 coverslips, 1 flask/pair 6-well dishes, sterile coverslips
11/4 Th view NLS-GFP cells from Drew's lab, IF PFA
rabbit anti 5mC
rabbit anti-DNMT1
goat anti-rabbit green (try red next time)
11/9 Tu
11/11 Th Veteran's Day
11/16 Tu
11/18 Th
11/23 Tu Th schedule
11/25 Th Thanksgiving
11/30 Tu
12/2 Th
12/7 Tu

Cell passaging

Cell passaging kdorfman Wed, 09/29/2021 - 17:07

Students split a flask of B16s into a new flask and a coverslip-bottom dish for viewing next class

  • 1 flask of B16-30 (wild type) per pair (at least 50% confluent)

  • 1 flask, 1 coverslip-bottom dish per student

  • 1 50 mL tube of DMEM per pair

  • 1 15 mL tube of PBS per pair

  • 1 0.5 mL aliquot of trypsin per pair

  • 5 mL serologicals

  • sterile transfer pipettes

  • 70% ethanol spray bottles

  • lots of gloves

  • handout

Lab 1 prep

Lab 1 prep kdorfman Wed, 08/25/2021 - 16:09
no. treated with conc [stock] time
10 untreated
2 VPA (valproic acid) 1 4 uM 20 uM over night
4 nocodazole 2 100 nM 1 mM (make 1uM 1st) overnight
2 trichostatin A 3 200 nM overnight
4 Cytochalasin D 4 250nM 10 mM 1 hour
2 Latrunculin 5 1 uM 1 mM 1 hour (in serum free medium!)

  1. Get VPA from Drew. 5X final conc.
    Add 0.5625 mL to each well containing 2.25 mL medium. ↩︎

  2. Add 97 uL DMSO to the 3 uL in tube to make 100 uL 1mM.
    Make 2mL 1uM in medium (2 uL 1mM + 1.998 mL medium).
    Add 0.25 mL to the 2.25 mL in each well. ↩︎

  3. TSA 10 mM from freezer.
    Make 1 mL 10 uM (1 uL TSA + 999 uL medium).
    Add 45 uL to each well. ↩︎

  4. Add 90 uL DMSO to the tube(makes 1 mM)
    Dilute this 1:1000 into medium (makes 1uM) (5X)
    Add .256 mL to each well ↩︎

  5. Mix 5 uL into 5 mL serum free medium (can use Fluorobrite)
    Replace the medium in each well with 2.5 mL of Latrunculin-medium mix ↩︎