2023 Fall Stephens
2023 Fall Stephens kdorfman Wed, 07/19/2023 - 14:34day | date | topic | prep |
---|---|---|---|
Tu | 9/5 | Learn your microscope | 10 HeLa parentals, FITC tubulin, 568 phalloidin, DAPI |
Th | 9/7 | Transmitted light and alignment | |
Tu | 9/12 | Cameras and calibration | |
Th | 9/14 | Numerical Aperture and resolution | |
Tu | 9/19 | Fluorescence | |
Th | 9/21 | Fluorescence photobleaching | (10 brand new triple stained HeLa slides?) Drew says no |
Tu | 9/26 | Immunofluorescence procedure | 20 coverslips of fixed HCT116 WT cells (grown on McCoy's) (maybe half treated with a drug the day before fixation), laminA, green secondary alpha tubulin, red secondary |
Th | 9/28 | Immunofluorescence imaging | |
Tu | 10/3 | Live cell imaging and cell culture | Split & seed another dish, live cell staining & imaging nuc blue, mitotracker red |
Th | 10/5 | Plasmid prep. and cell culture | Eb1 in liquid culture, Zymo miniprep |
Tu | 10/10 | Monday schedule | (Kate transfects HCT116's with Eb1 plasmids, seeds on viewing dishes) |
Th | 10/12 | Transfection imaging / Makeup day | image kate-transfected cells |
Tu | 10/17 | Midterm | Mai does trial runs |
Th | 10/19 | FLEXIBLE DAY / CURE intro | |
Tu | 10/24 | 1st day CURE | Students fix & stain coverslips |
Th | 10/26 | ||
Tu | 10/30 | ||
Th | 11/1 | ||
Tu | 11/6 | ||
Th | 11/8 | ||
Tu | 11/13 | ||
Th | 11/15 | ||
Tu | 11/20 | ||
Th | 11/22 | THANKSGIVING | |
Tu | 11/28 | ||
Th | 11/30 | Final Project due | |
Tu | 12/5 | Neurobiology | |
Th | 12/7 | Neurobiology |
09/26/23 Immuno procedure
09/26/23 Immuno procedure kdorfman Thu, 09/21/2023 - 21:19Immunofluorescence Procedure Tu 9/26/23
Materials
- PBS-Tw-Az for rinsing
- Little beakers
- forceps
- enough for 4 humid chambers
- petri dish
- filter paper
- parafilm
- fixed coverslip of HTC116 cells, 1 per student
- Mixed primary antibodies
- Mixed secondary antibodies
- Goat anti mouse 488 (green)
- Goat anti rat TRITC (red)
To mix primaries:
- Lamin A:
- Use 4 aliquots of 5 µL each
- Add 125 µL PBS-Tw-Az-BSA to get (slightly less than) 2X strength
- total volume: 520 µL
- Alpha Tubulin:
- Use 3 aliquots of 4 µL each
- Add 200 µL PBS-Tw-Az-BSA to get 2X strength
- total volume = 600 µL
- Mix 1:1, and aliquot ~104 µL per pair. Make 10 aliquots
To mix secondaries
- Green anti-mouse:
- 2 1µL aliquot
- 300 µL PBS-Tw-Az-BSA
- total volume: 602 µL
- Red anti-rat:
- 2 5 µL aliquots
- 260 µL PBS-Tw-Az-BSA each
- total volume: 530 µL
- Mix 1:1, and aliquot ~106 µL per pair. Make 10 aliquots
10/03/23
10/03/23 kdorfman Mon, 10/02/2023 - 20:54Live Cell Staining & Imaging
Students use the dish they seeded previous Thursday.
Need "staining solution":
- Fluorobrite
- Nuc Blue
- Mitotracker Red
10/10/23
10/10/23 kdorfman Mon, 10/02/2023 - 19:38Kate transfects HCT116 cells with Eb1 plasmids.
Need 8-10 viewing dishes, seeded with transfected cells 1 to 2 days before viewing.
1 reaction needs ~ 106 cells/mL
100 uL cells per reaction (barely 8 drops)
So do 2 reactions, each with 106 cells.
2 days before, seed a 25 cm2 flask with 0.5 106cells.
(4 days before, seed with 0.125 106 cells)
1 drop of the 100 uL reaction into its own dish.
On transfection day, divide into 2 reactions
Notes on actual protocol followed:
Friday 10/6
- Start with 2 confluent 12.5 cm2 flasks
- (~1.25 x 106 cells)
- Need 1-5 x 106 cells/mL on Monday
- Split 1/8 to allow for 3 doublings
- Stop reaction with 1.6 mL
- Seed 0.2 mL into each of 2 new flasks.
Monday 10/9/23
- Put all the cells from one confluent 12.5 cm2 flask
- should be confluent again
- into a new 25 cm2 flask
Tuesday 10/10/23
- There should be 2.5 x 106 cells
- Trypsinize
- Stop reaction with 2 mL
- split suspended cells into two microfuge tubes.
- Spin down, and proceed with 2 Mirus nucleofection reactions
- Make 2 12.5 cm2 flasks
10/17/23
10/17/23 kdorfman Mon, 10/16/2023 - 19:26Trial run for Lamin antibodies
Fix 15 min in paraglut
1o Ab: Rabbit anti-lamin B
- dilute 1:1000 with PBS-Tw-Az-BSA
- 2 µL antibody into 2 mL buffer
- 1 hr 37C
2o Ab: goat anti-rabbit
- 1 hr RT incubation
Cell type | # coverslips |
---|---|
MEF WT NLS-GFP (40) | 3 |
HCT-116 WT (60) | 3 |
DU-145 (74) | 4 |
Stain with Hoechst (1:10,000) in the final PBS-Tw-Az rinse
Mount with non-DAPI Fluoromount (Fisher OB100-01)
10/24/23
10/24/23 kdorfman Mon, 10/16/2023 - 19:36Students begin projects
Kate provides fixed coverslips (seeded 10/22, fixed 10/24/23)
Cell line | medium | variant | treatment | no. coverslips |
---|---|---|---|---|
HCT116 | McCoy | WT (60) | 4 | |
LB1-AID-GFP (68) | 2 | |||
MEF | DMEM | WT (39) | control | 6 |
VPA | ||||
DZNP | ||||
LMNB1-/- (02) | 3 | |||
DMEM+G418 | LA KD (37) | 3 | ||
Prostate cancer | RPMI-1640 | DU145 (74) | 2 +3 from Mai | |
RPMI-1640 | LNCaP (77) | 2 + 3 from Mai | ||
DMEM | PC3 (76) | 2 |
Materials:
- Paraglut fix (enough for 30 wells)
- PBS to rinse 30 coverslips
- PB-TW-Azide (enough for 24 dunking beakers and 30 wells)
- 1o antibodies
- mouse anti-NP (nuclear pore)
- mouse anti-LB1
- rabbit anti-LB2 ab151735 (get one 10 µL aliquot from Drew - dilute 1/1000)
- 2o antibodies
- goat anti-mouse green for NP
- goat anti-rabbit red for LB1 & LB2
- Slides
- slide labels
- DAPI & droppers
- forceps
- dunking beakers
- humid chamber materials
Project | people | scope | IF coverslips | IF details |
---|---|---|---|---|
MEF VPA | Olivia & Juliana | 1 | 1x 39 unt, 1X 39 VPA, another VPA if pos | standard NP LB1 IF |
MEF DZNep | Karan & Nate | 2 | 1x 39 unt, 1X 39 DZNep, another if pos | standard NP LB1 IF |
MEF LMNB1-/- | Cole & Grif | 3 | 1X 39, 2X 02 LMNB1-/- | 39 & 02 NP LB2 IF, 1x 02 NP LB1 IF |
MEF LA KD | Shrushti, Emma, Anna | 8 | 1X 39, 2X 39 | standard NP LB1 IF |
HCT116 LB1 live 1 | Sam & Jillian | 5 | 68, 1x unt & 1x VPA | LB2 IF only |
HCT116 LB1 live 2 | Isabelle & Jason | 6 | 60, 1x unt 1x VPA; 60, 1x unt 1x VPA | one set NP LB1 IF; other set NP LB2 IF |
Prostate Cancer 1 | Tyian & Antonia | 4 | 2x 74, 1x 76 | standard NP LB1 IF |
Prostate Cancer 2 | Kelsey & Pedro | 7 | 2x 74, 1x 77 | standard NP LB1 IF |