10 slides w/ DAPI

(1 per microscope + 2 extra)

Micrometers

5 or 6 slides each:

• Rhodamine Phalloidin "1G"
• Stained for rat anti-tubulin + goat anti-rat FITC (or GFP-tubulin cells) "2B"
• Acridine orange stained for nucleoli "3G"
• Stained with with mouse anti-Golgi + goat anti-mouse 568 "4G" (formaldehyde fixed)
• Stained with rabbit anti-ZO1 + goat anti-rabbit Cy (cell-junction) "5G"

20 fixed coverslips for phalloidin staining

3 MatTeks per group for mitotracker and ?

aliquots of

• phalloidin
• mitotracker
• non-CO2 medium

Finish looking at slides, discuss cell division

10 slides DAPI + tubulin

2 MatTeks per group each:

• LLCPK with GFP tubulin
• LLCPK parentals

NUC-Blue for staining chromosomes in live cells (Invitrogen R37605 or Fisher NC0291762), 1 bottle per station

• Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

Non-CO2 medium

4 MatTeks per group of LL-GFP-tubulin

Double-strength inhibitors, made up in non-CO2 medium:

• STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

• need 1 mL aliquots 10 µM in non-CO2 medium for students

• Make 100 mM stock:

  • 1 g in bottle.
  • Mix with 27.5 mL DMSO
  • heat to 65C (~1 hr), vortex
  • if necessary, filter sterilize to remove insoluble particles.
  • save as 1 mL aliquots. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)

• Make 1 mM stock from the 100 mM stock

  • 10 µL 1mM stock
  • 990 µL DMSO
  • vortex
  • it will crystallize in the refrigerator. Warm and vortex to redissolve it.

• Make 17 mL 10 µM in medium for students

  • 170 µL 1 mM stock
  • medium to 17 mL
  • aliquot

• Make 10 µL aliquots of 1 mM stock

  • label says to add 990 µL medium to make 10 µM working solution

from Alyssa:madke STLC in DMSO and that she heated to 42C, and vortexed. She says it went from clear to yellow/brown. we use the reagent at micromolar - 1-10 µM. Her stock solution was 1mM.

students will be adding 1 mL of inhibitor to 1 mL medium in dish; the ready-to-use 1 mL (in medium) should be at least 2µM

• Taxol (20 µM final)
• soluble in DMSO, not water.
  • students pick either Taxol or nocodazole, so make only 10 each
  • each tube has 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)
  • add 490 µL to make 200µM in two tubes!
  • mix with medium in 15 mL conical to make 10 mL at 20 µM

• Nocodazole (3.3 µM final)

  • each tube has 3 µL 33mM
  • add 97 µL to tube to make 100 µL of 1 mM
  • 33 µL 1mM + 10 mL medium = 3.3 µM

• Nuc Blue from Invitrogen (or Fisher NC0291762), 1 bottle per station

  • Remove from culture dish after ~10 minutes. Replace stain medium with fresh medium. Otherwise it gets brighter and brighter as movie goes on

Myosin & tubulin expressing cells

2 MatTeks each per group

For ML-7, the final conc should be 75uM, so the 2X needs to be 150uM. For the Jasplak: the final conc should be 7uM so the 2X needs to be 14uM (but if 15 is easier, go with it). we use latrunculin at 5uM, and I think cytoD should be similar (I think I previously gave a range for cytoD of 1-10uM). Let's go with 5, so the 2X would be 10uM.

Inhibitors in non-CO2 medium (1 mL aliquots) each group does one (so make 5 of each):

• cytochalasin D Sigma C2618 or C8273

  • tubes have 40 µL 2.5 mM
  • label says add 460 µL to make 500 µL of 200 µM
  • need 10 µM (2x final working concentration)
  • 20 µL 2.5 mM
  • 5 mL non0CO2 medium
  • gives 5 1 mL aliquots at 10 µM

• Jasplakinolide – Santa Cruz biochemical, product sc202191 F

  • inducer of actin polymerization & stabilization
  • F-actin probe
  • MW = ~710
  • powder is stable frozen over a year
  • stock is stable 3-4 mo at -20C
  • Make 1 mM stock with 50 µg in 70 µL DMSO
  • Add 5 mL non-CO2 medium to make ~14 µM
  • 50 nM to 5 μM working range
  • minutes to hours incubation time

• ML-7 Sigma, 12764.

  • soluble 10mg/mL in 50% EtOH
  • MW = 452.74
  • 5 mg in bottle
  • add 1100 µL to make 10 mM solution
  • used at ~50 µM (1 µL/1mL) (Pat says 75 µM)
  • 75 µL 10 mM + 5 mL non-CO2 medium makes 150 µM
  • make 15 µL aliquots of 10 mM. Add 985 µL medium to make 150 µM

Students limited to 3 MatTeks of one of the following:

• GFP tubulin
• tubulin-myosin cells

Plus their choice of inhibitors:

• latrunculin
• taxol
• nocodazole
• jasplakinolide
• ML-7
• STLC

Report on mitosis experiments

• no. 5 forceps
  • Fisher S95311 Straight; Ultra fine pointed tips; Aven Tools 18056ER $12.53
  • Digikey 243-1099-ND Aven Tools 18062-ER $15.65
  • Digikey 243-1054-ND Aven Tools 18062EZ $5.75 (green)
  • 243-1051-ND 18056EZ (turquoise) sharpest!
  • 243-1053-ND 18013EZ (yellow)

Forceps tip sizes

• methyl cellulose 3% ~200 mL > Flinn Scientific M0171 $7.55 100 mL

It takes ~ a week of stirring to make the powder ( Fisher S80080 100G) go into solution at this concentration!

• Fish water (get a carboy full from Craig)

• Lithium Chloride (MW = 42.4) solutions:

  • 10M LiCl in 10 mL H2O (4.24 g) (vortex in 15 mL conical. gets hot.) Check stock solutions first

  • 0.3 M LiCl in fish water (1.272 g/100 mL)

  1 µL 10 M/1 mL fish water

• deep depression slides (Pyrex 9-spot plates)

• 60 mm petri dishes - CellTreat with gripping ring are good for this (sterility not necessary)

• glass-bottom 29 mm petri dishes (InVitro Scientific)

• 2 packs (2 x 48) Netwell baskets (Corning 3477)

Fisher 07-200-211 $215.55

• 12 well plates (for the baskets to fit in)

• timers

• superglue

• short tip pasteur pipettes

• fishing line

• transfer pipets (large, non-sterile)

• mirror base dissecting microscope

https://wahoo.cns.umass.edu/node/723 NM

• Inoculate overnight culture in LB + antibiotic (TET for HT115(DE3) strain) (2-5ml). Shake overnight at 37C.

• Inoculate 25 ml LB + antibiotic with overnight culture, 1:100 dilution. Grow cells to OD595= 0.4. Can grow cells in 50 ml sterile centrifuge tube.

• Spin cells 10 min 3000 rpm at 4C.

• Resuspend pellet in 0.5X original volume cold, sterile 50 mM CaCl2 (12.5ml). Resuspend by GENTLY pipetting up and down a few times with a wide bore pipet--no vortexing.

• Incubate on ice 30 min.

• Spin as before at 4C.

• Resuspend pellet as before in 0.1X original volume CaCl2 (2.5ml). Keep cells cold (4C).

• Use 50-200 ul for transformation. Cells can be used as is for up to three days (stored at 4C).

• The cells can be frozen by: adding glycerol to final concentration of 10%, rapid freezing on dry ice/EtOH, and storing at –80.

Cloning Bootcamp

• 37C heatblock
• 42C heatblock
• Age1
• 10X buffer NEB1.1
• rSAP
• Sterile water for cloning
• Ligase + 2X rapid lligase buffer
• 1 sample: PCR cleanup kit (Qiaquick) (one per group)
• 1 tube (5 rxns) Competent cells (DH5 alpha, just for cloning)
• 5x LB+Amp plates
• Sterile beads for spreading
• 30 ng/ul backbone plasmid for cutting (estimated 15n/ul after elution w/ 30ul)
• Gel purified inserts (gamma tub, centrin, sas-6 at 15ng/ul)
• LB (plain) for transformation recovery

Naegleria 101 (2/7/22)

Activities:

• Differentiate cells,
• watch amoebae (phase microscopy),
• look at flagellates.
• Pick colonies for cloning.

Materials:

• 4 LB+ Amp +
• Tubes for growing cultures from previous lab’s cloning
• Toothpicks for above

8 QIAprep Spin miniprep kits for 4 cultures each

Notes before starting

• Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
• Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C.
• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge.

Each kit contains:

• 4 spin columns
• 6 buffers, as below

Other materials

• Age1 + cutsmart
• 8 2mL tubes of LB broth
• competent HT115 cells from -80
• two 1% gels for wide gel rig, 2 small-well combs each
  • 2 g agarose
  • 200 mL 1X TAE
  • 20 uL SYBR Safe
• 1X TAE running buffer
• 1KB DNA standard

Equipment

• Microcentrifuges capable of 13000rpm
• Hot blocks at
  • 37
  • 42
• two wide gel rigs
• two power supplies
• two blue light boxes with photography box

M Feb 14

32 (?) tubes of LB-amp

*4 PCR machines with dual blocks - enough for eight groups of students

*8 DNA gel apparatus

*3 weighing balance for pipetting exercise

*10 mM dNTPs, 60 microliters total

*Phusion DNA polymerase, 10 microliters total (we prefer Phusion from NEB because we are amplifying with 80 nt long primers. I don’t recall if your freezer has any of this DNA polymerase. If not, I’m willing to just use the one in my lab)

For Thursday, Jan 29th:

MBOMS will run gel and need to coordinate with other labs to use the gel doc.