2020 remote learning lab
2020 remote learning lab kdorfman Fri, 08/07/2020 - 15:421 - Pollen
1 - Pollen kdorfman Fri, 08/07/2020 - 15:43Make LPGM
with 5%, 7%, and 10% sucrose
Incubate briefly in LPGM in a 2 mL tube in the Ferris wheel, at a slow spin speed, parallel to the direction of rotation so the tube goes upside down and the liquid moves from top to bottom of the tube.
Can image at 4x on a poly-lysine coated slide . Make a vaseline circle, put some pollen suspension inside it, cover with coverslip.
Set up time-lapse imaging: 20 images every 5 minutes. Find a field of view with several pollen grains in focus at once, all starting to germinate.
In a multiwell plate The surface of one well of a 12 well plate can be covered by as little as 400uL water.
Need to cover with poly-lysine. Already have ~50 mL of 0.1% working solution.
So can coat ~125 wells
Well diameter = ~22 mm
In a cover-slip bottom 60 mm dish
500 uL coats the well.
Coverslip diameter = 30 mm
How many unique fields of view can we get in one dish?
Pollen tubes grow ~0.2 um/sec = 12um/min = 0.012 mm/min
magnification | field diam (mm) | field diam (um) |
---|---|---|
4x | 5 | 5000 |
10x | 2.2 | 2200 |
40x | 1.1 | 1100 |
Reasonably good optics at 10x; camera adds magnification.
Coat all wells on 3 12-well plates, one for each concentration of sucrose.
2 - Molecular Biology
2 - Molecular Biology kdorfman Fri, 08/07/2020 - 15:43- H2B-mCherry (Addgene 20972) (Kan resistant)
- ecadherinGFP (Addgene 28009) (Kan resistant)
Restriction Enzymes
- PvuI-HF (cut smart)
- BamHI-HF (cut smart)
- KpnI-HF (cut smart)
A "Typical" Restriction Digest
Reagent | amount |
---|---|
Restriction Enzyme | 10 units is sufficient, generally 1 µl |
DNA | 1 µg |
10X NEBuffer | 5 µl (1X) |
Total Reaction Volume | 50 µl |
Incubation Time | 1 hour |
Incubation Temperature | Enzyme dependent |
Gel Loading
- 1 % agarose in TAE
- 1/10,000 SYBR Safe
- 20 wells
- wide gel
- 10 uL/well
Lanes 1, 10, 20 have NEB 1Kb ladder mixed in this ratio
- 1 uL ladder
- 1 uL loading dye
- 6 uL water
Tube | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
---|---|---|---|---|---|---|---|---|
Ingredient | uncut control | BamH1 | KPN1 | Pvu1 | BamH1 + Kpn1 | BamH1+Pvu1 | Kpn1+Pvu1 | BamH1+Kpn1+Pvu1 |
10X NEBuffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
plasmid 20972 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
BamH1 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 1 |
KPN1 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 1 |
PVU1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 |
water | 42 | 41 | 41 | 41 | 40 | 40 | 40 | 39 |
Tube | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 |
---|---|---|---|---|---|---|---|---|
Ingredient | uncut control | BamH1 | KPN1 | Pvu1 | BamH1 + Kpn1 | BamH1+Pvu1 | Kpn1+Pvu1 | BamH1+Kpn1+Pvu1 |
10X NEBuffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
plasmid 28009 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
BamH1 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 1 |
KPN1 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 1 |
PVU1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 |
water | 46 | 45 | 45 | 45 | 44 | 44 | 44 | 43 |
Restriction sites
Plasmid | Bam1 | Kpn1 | Pvu1 | plasmid length |
---|---|---|---|---|
20972 (H2B) | 1724 | 2117 | 3567 | 6476 |
28009 (ecadherin GFP) | 3703 | 3059 | 6007 | 8820 |
Digest fragments
Plasmid | BamH1 + Kpn1 | BamH1+Pvu1 | Kpn1+Pvu1 | BamH1+Kpn1+Pvu1 |
---|---|---|---|---|
20972 (H2B) | 393, 6083 | 1843, 4633 | 1450, 5026 | 393, 1450, 4633 |
28009 (ecad) | 646, 8174 | 2304, 6516 | 2948, 5872 | 646, 2304, 5870 |
3 - Bacterial growth
3 - Bacterial growth kdorfman Fri, 08/07/2020 - 15:44Grow bacteria
Streak frozen cells on agar plate
Pick a colony and grow overnight in liquid LB (plus antibiotic if necessary)
Concentrate ON culture in centrifuge
Make 2 dilution series: 10-fold, 2-fold
2 fold
- tubes 1A - 10A
- 0.5 mL LB in each
- add .5 mL culture to first, mix thoroughly
- serially dilute by moving 0.5 mL along the series*
10 fold
- tubes 1B - 10B
- 0.9 mL LB in each
- add .1 mL culture to first, mix thoroughly
- serially dilute by moving 0.1 mL along the series
Take OD measurements
- Load 250 uL of each dilution into 96 well plate
- samples in 1st 10 wells of 1st 2 rows
- LB in last 2 wells
- Read, using bug OD
Plot results
- Put fraction of original culture next to each OD
- Cut, paste special, values only
- Sort data
- Make a scatterplot
- Find the lowest concentration just above the noise for hemocytometer
Hemocytometer
- Load 10 uL into hemocytometer.
- Photograph.
- Also take photographs of too concentrated and too dilute samples
Colony Counts
- spread 100 uL from last 5 10-fold dilutions on agar plates
- Grow overnight
- Photograph
4 - Lac Operon
4 - Lac Operon kdorfman Fri, 08/07/2020 - 15:45Qualitative lac operon experiment
Grow GFP E. coli in different sugar media, then photograph in a 12 well plate on a blue light box through the orange filter.
Streak GFP E. coli on an LB-glucose-kan plate, grow overnight
Pick a colony, grow in overnight in liquid LB-glucose-kan (to prevent it turning green)
Spin down the ON culture (take a picture of the pellet for other use!)
Rinse several times (to get rid of the glucose)
Fill 12 well plate as indicated below. 2 mL per well:
additive | none | IPTG 0.01mM | lac 0.25M | gal 0.25M |
---|---|---|---|---|
none | ||||
glu 0.25M | ||||
mal 0.25M |
- Photograph
- in white light
- in blue light
Read in plate reader for blanks (OD and FL)
- 12-well plate is too tall for reader!!!
- Sample 100 uL of each to read blanks (OD & FL in a 96 well plate)
Inoculate each well with a small amount of culture.
Incubate on the shaker.
Photograph
- in white light
- in blue light
- Read in plate reader (OD and FL)
- Take 100 uL (?) samples to read in 96 well plate
Media
Need enough for the qualitative experiment ~5 mL each
Need enough for the quantitative experiment: 96 well plate @ 250 uL/well = ~25 mL total
Make 25 mL of each high concentration stock; mix the combinations from that
Add 50 µg/mL Kanamycin as indicated here.