Make 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.

Bacterial plasmids:

• 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

Molecular biology PowerPoint from 2020 remote class

Grow 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

Part 1 of PowerPoint from 2020 remote lab

Part 2 of PowerPoint from 2020 remote lab

Qualitative 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.

Lac Operon PowerPoint from 2020 remote class