For 9/2 & 9/7 2021
For 9/6 & 9/8 2022

Lab 1.1 protocol

• Pipetters & tips
• trash bucket
• Balances
• water beaker
• liquid waste bucket
• gloves
• Fluorescein 1 mM (~5 mL)
• 12 glass tubes in rack
• white and black strips behind rack
• yellow filters
• blue flashlights

9/9 & 9/14 2021

Lab 1.2 protocol

• Getting files from Wahoo.

• Link to bcrc accounts

• tube to mix 2 mL

• rack to hold tubes: fluorescein stock tube, NaOH tube, dilution tube

• 1 mM fluorescein in 10mM NaOH

• 10mM NaOH

• 96 well fluorescence plate

• tape & marker

• liquid waste beaker

• tips & other dry trash beaker

9/16 & 9/21 2021

• tube to mix 2 mL

• rack to hold tubes: fluorescein stock tube, NaOH tube, dilution tube

• 1 mM fluorescein in 10mM NaOH

• 10mM NaOH

• 96 well fluorescence plate (can use saved plate)

• tape & marker

• liquid waste beaker

• tips & other dry trash beaker

• two unknown fluorescein concentrations (one too dilute for Abs, one too concentrated for FL)

9/16 & 9/21 2021 9/20 & 22 2022

2020 experiment

Bacterial plasmids:

• H2B-mCherry (Addgene 20972) (Amp resistant) (Labeled A)
• ecadherinGFP (Addgene 28009) (Amp resistant) (Labeled B) GROWS SLOWLY! ALLOW EXTRA TIME!

Miniprep 2022

• Tuesday: Zyppy (use up old aliquots)

• Thursday: ZR Classic

Mini-Prep 2021

• micro pipetters & tips
• labeled racks in fridge
• 1 mL each E. coli culture (concentrated by centrifugation) per pair
• centrifuges

• Miniprep kits - aliquot miniprep reagents per pair:

  • sterile microfuge tubes
  • 250 uL Buffer P1(+ RNaseA+LyseBlue)/rxn = 510 uL per pair
  • 250 uL Buffer P2/rxn = 510 uL per pair
  • 350 uL Buffer N3/rxn = 710 uL per pair
  • 2 QIAprep spin columns
  • 0.5 mL Buffer PB/rxn = 1.1 mL/pair
  • 0.75 mL Buffer PE/rxn = 1.51 mL
  • 50 uL Buffer EB (10 mM Tris-Cl pH 8.5) per rxn = 110 uL per pair
  • sterile water

Restriction Digest

9/27 & 9/29 2022 (9/23 & 9/28 2021)

2020 experiment

Reagents & Materials

• 2 plasmids from Lab 2.1

• Wide format gel

  • 6 1% 100 mL SYBR Safe gels
  • TAE in carboy
  • 2 20-well combs per gel (buy more!)
• Restriction enzymes (get the large size!)
  • BamH1-HF
  • Kpn1-HF
  • Pvu1-HF
• Diluent B to dilute enzymes to 1/4 strength
• NEB Purple 1 Kb ladder
• Purple loading dye SDS-free (comes with enzymes)
• hot blocks for 168 reactions (!) Maybe an incubator?

Restriction Digest Volumes

Aliquots

16 reactions per pair (8 reactions x 2 plasmids):

• 3 single
• 3 double
• 1 triple digest
• 1 uncut

• For 3 single, 3 double, 1 triple digest, and 1 uncut x 2 plasmids per pair:
  • Cut smart: 50 µL (16 rxns x 2.5 µL = 40 µL)
  • Enzymes: dilute to 1/4, then give 20? µL (4 rxns x 4 µL = 16 µL)

Restriction sites

Digest fragments

9/30 & 10/5 2021

10/7 & 10/12 2021

Wet materials:

• LB plates, 8 per pair = 96/section
• LB broth (cold) ~10 mL/pair
• E. coli at stasis: 3 mL aliquots in round bottom tubes, in fridge, 1 per pair = 36 mL
• ice in small container

Equipment

• hemocytometers
• glass beads
• beaker for used beads at each table
• tips beaker
• liquid waste beaker
• microfuge tubes & rack
• marker
• clear 96 well plate
• pipetters & tips

Plate reader program

• BUG_OD
• 1st 2 rows

10/14 & 10/19 2021

Give them the Excel growth curves.

Consider doing a 37C growth curve as well.

10/25 & 10/27 2022: Combined with 4.2

Make 1 set of 12 cultures for each table.

10/21 & 10/26 2021

• sterilize 12 small flasks for cultures
• sterilize at least 288 glass culture tubes

See prep for 2015

See LB sugar recipes

• Make 50 mL of each mixture
• Put 25 mL into flask for shaking incubator
• Put 1 mL into microtube for blanks.
• Save the rest for the second section (in case you need to grow more cells).
• Make 12 (or 6) 2 mL aliquots - one set per group; 5 mL aliquots of LB

See E. coli for Lac operon

Spread glucose + Kan on plain LB plate, then

Streak incubate overnight, and pick a colony into each medium.

Allow to grow, but not too long! Over day, but not over night? Overnight is OK, but with temp below 30C

Put out blue flashlights and yellow filters

Class can share one black fluorescence plate

10/28 & 11/2 2021

Make 12 tubes of 12 media, 5 mL each.

6 sets per day.

Day before:

• make LB-glu (mix 20% glucose from genetics 1:1 with LB if necessary)
• Start overnight culture in LB-glu

Day of:

• Borrow Biochem multi-channel pipet plus small tips. Dial down to 5 uL
• Subculture into LB glu again
• Just before use, wash with LB 3x
• Resuspend in 10 mL LB
• Need OD = 0.02 in the wells
  • Need 10 mL to put in sterile petri dish to fill pipet
  • Need an OD that doesn't appreciably raise the OD of 205 uL LB (OD =~0.2)
  • Aim for blank corrected OD of 0.8
  • Dilute as necessary

11/4 & 11/9 2021

11/18 & 11/30 2021

11/18 Thursday

Group 1: Botros et al.

• Zone of inhibition
• E. coli Addgene 20972 from molecular biology experiment
• LB Amp 25 mL
• alcohol (ethanol or isopropanol?)
• disinfectant??
• 20 LB amp plates for zone of inhibition
• sterile discs
• sterile forceps

Group 2 (Vengalil et al.):

• E. coli Addgene 20972 from molecular biology experiment grown in LB-amp
• LB amp salt-free
• LB amp 2M NaCl
  • add 2.922 g NaCl per 25 mL salt-free LB
• LB amp 2M KCl
  • add 14.9 g KCl per 100 mL LB
• 20 LB amp plates

Group 3 (Long, et al.):

• 40 LB plates (no antibiotic)
• Replica plate materials
• Kid's hand sanitizer
• room swipe access (need names and Spire IDs)

Group 4 ( Lee, et al.):

• E. coli GFP culture in LB Kan
• LB Kan ~25 mL
• LB-glu Kan ~15 mL
• 96 well black plate, shared with group 5
• rewrite script for only the rows they use

Group 5 (Creto, et al.)

• E coli GFP culture in LB Kan
• LB Kan ~25 mL
• LB-glu Kan ~15 mL
• LB-lac Kan ~15 mL
• LB-gal Kan ~15 mL
• 96 well black plate, shared with group 4
• rewrite script for only the rows they use

11/30 TUESDAY

McKnight, Carson, Matfes, Berke

• change pH of LB
• GFP expression
• plate reader

Bowen, Howall, Abraham, Wald

• fertilizer - N-based vs P-based.
• Nitrogen vs phosphorus fertilizer
• LB,
• sterile tubes,
• plates
• Culture (non-GFP?)

Boyd, Ebian, Melusen, Manning

• pH again

Cloutier, Dawiczyk, Tan, Joyce

• mouthwash
• plate reader
• culture (non-GFP?)
• Liquid LB

Alexander, Ferriera, Beinstein, Birnbach

• comparison of various substances at killing E. coli
• LB broth and plates
• E coli culture (not GFP?)

Crotty, Galligan, Horrigan, Jansen

• NaCl
• KCl
• plate reader overnight
• agar plates

11/23 (Thursday section) & 11/30 2021

12/2 & 12/7 2021