Lab prep, media, and supplies


Introduction

This information is primarily for the course TAs and media prep person, but might be of some interest to the students. All amounts listed below are based on a class of 40 students in 2 lab sections.


Media Schedule

TENTATIVE - Check weekly against supplies.

Deadline Media Type Number Make
Jan 18 PYD plates 50 1.5 liters
BIM tubes 50 1.5 liters
E tubes 50 1 liter
YPD-Amp tubes 50 0.5 liter
NA plates 100 3 liters
NAM tubes 50 0.5 liter
Jan 25 PYD plates 50 1.5 liters
BIM tubes 50 1.5 liters
YPD-Amp tubes 50 0.5 liter
NA plates 100 3 liters
NAM tubes 50 0.5 liter
Feb 01 PYD plates 50 1.5 liters
BIM tubes 50 1.5 liters
BIM plates 50 1.5 liters
E tubes 50 0.5 liters
YPD-Amp plates 50 1.5 liters
YPD-Amp tubes 50 0.5 liter
NA plates 100 3 liters
NAM plates 50 1.5 liters
NAM tubes 50 0.5 liter
Feb 08 PYD plates 100 3 liters
BIM plates 50 1.5 liters
E tubes 50 0.5 liter
YPD-Amp plates 50 1.5 liters
NA plates 100 3 liters
NAM plates 50 1.5 liter
Feb 15 BIM plates 50 1.5 liters
E-agar plates 50 1.5 liters
PYD plates 50 1.5 liter
YPD-Amp plates 50 1.5 liters
NAM plates 50 1.5 liters
Feb 20 BIM plates 50 1.5 liters
YPD-Amp plates 50 1.5 liters
E-agar plates 50 1.5 liters
BIM plates 50 1.5 liters
NAM plates 50 1.5 liter
Mar 01 BIM plates 50 1.5 liters
E-agar plates 50 1.5 liters
MarĀ 08 NA plates 150 4 liters

Media preparation

PYD plates

Add to 1 liter of distilled water:

  • 2g peptone
  • 2g yeast extract
  • 5g dextrose
  • 15g agar

Mix & autoclave. Cool to about 50C, mix, & pour into plates

YPD-Amp

Add to 1 liter of distilled water:

  • 20 grams dextrose
  • 10 grams peptone
  • 5 grams yeast extract

Adjust to pH 5.0 using dilute HCl

For tubes dispense 10ml into each of about 100 16x150mm tubes, cap (USE YELLOW CAPS) & autoclave. Allow to cool, and add 40ul of 25mg/ml ampicillin to each tube. This ampicillin stock can be made by mixing 0.25g ampicillin-HCl to 10ml distilled H2O, dissolve, and filter sterilize (store frozen).

For plates, add 15g agar per liter, mix, and autoclave. Cool to 50C, then add 0.1 gram ampicillin powder, mix and pour into plates

BIM

Add to 1 liter of distilled water:

  • 1 gram (NH4)SO4
  • 0.5 gramK2HPO4
  • 0.2 gram MgSO4
  • 2 grams NaCl
  • 5 grams NaHCO3
  • 1.5 grams Yeast extract
  • 1.5 grams Glycerol

Adjust pH to 7 w/ dilute HCl or NaOH

For tubes, autoclave and dispense 20ml into each of about 50 sterile 25ml Balch tubes (use aluminum foil to cover the tubes for autoclaving and storage after the media is added)

For plates, add 15g agar, mix & autoclave. Cool to about 50C & pour into plates

E-agar

E media is just artificial sea water. Add 30cc of Instant Ocean powder per liter of tap water.

For best results, use spent marine aquarium water or ocean water instead.

For tubes, put 10ml of sea water into 16x150 culture tubes, cap & autoclave (USE GREEN CAPS). Add "some" agar powder to each tube just before use.

For E-agar plates, add 20g agar to 1 liter E media, autoclave, mix & pour.

NA (nutrient agar) plates

Nutrient agar is supplied in pre-mixed form - prepare as directed

Note that PYD (above), TSA (trypticase soy agar), YT, LB, or any other basic rich agar can be substituted for NA for the purposes of this class.

NAM media

Nutrient agar for preparing this media (Nutrient Agar/mineral water, or NAM) is supplied in pre-mixed form - prepare as directed. The only difference is that instead of distilled water, use Pelligrino mineral water.

Note that PYD, TSA (trypticase soy agar), YT, LB, or any other basic rich agar can be substituted for NA for the purposes of this class. Likewise, and high mineral content mineral water can also be substituted.

For liquid media - dispense & autoclave

For plates - add 15g agar, mix, and autoclave. Gently mix and pour into plates as usual.


Student Drawer Material

  • slides (box)
  • cover slips (box)
  • immersion oil
  • Kimwipes (box)
  • eye protection
  • fine tip sharpie
  • microfuge tube rack
  • 1ml blue pipet tips (rack)
  • 200ul yellow pipet tips (rack)
  • 1.5ml microfuge tubes (box)
  • 0.5ml microfuge tubes (box)
  • hockey stick
  • EtOH bottles w/ EtOH for flaming
  • dropper bottle w/ water
  • forceps
  • loop
  • innoculating needle

Student Station Material

  • trash container
  • sharps disposal box
  • Centrifuge bottle rack (for Wino columns)
  • Green tube rack

Equipment

  • 24 Phase-contrast microscopes
  • 3 heating blocks
  • 3 vortexes
  • 24 P20's
  • 24 P200's
  • 24 P1000's
  • 3 minigel units, tray & combs
  • 3 power supplies
  • 1 photodocumentation system
  • 3 microfuges
  • 1 incubator at 30C
  • 3 30C incubator with fluorescent lights
  • 6 anaerobe jars
  • 1 thermocycler

Supplies & Chemicals

Item Need Quantity Vendor Cat # Price Comment
Supplies            
Petri dishes 2 case of 500 Fisher 08-757-14G 70.07 from stock
plastic cylinders for Wino columns 55 8' lengths Home Depot     1 foot segments of fluorescent light covers
#9 stoppers 60 5 lbs (~40) Fisher 14-130N 14.01  
Balch tube bungs 150 case of 100 Bellco 2048-11800 51.85  
25ml Balch tubes 150 case of 100 Bellco 2048-00150 164.25  
16 x 150 culture tubes 1 case of 1000 Fisher 14-961-31 45.62 stockroom
red caps for 16mm culture tubes 200 case of 1000 Fisher 14-957-92D 43.77 stockroom
yellow caps for 16mm culture tubes 200 case of 1000 Fisher 14-957-92F 42.21 stockroom
green caps for 16mm culture tubes 200 case of 1000 Fisher 14-957-92E 43.77 stockroom
200ul (yellow) pipet tips 12 case of 10 bx Fisher 02-681-155 31.36  
1000ul (blue) pipet tips 12 case of 10 bx Fisher 21-197-8J 42.09  
slides 5 case Fisher 12-550A 152  
coverslips 5 case Fisher 12-542B 82.68  
1.5ml microfuge tubes 500 pack of 500 Fisher 05-408-129 16.54  
0.5ml microfuge tubes 250 pack of 500 Fisher 05-408-120 16.08  
22g sterile needles 2 box case of 100 Fisher S80350A 14  
1ml sterile syringes (21g needles) 2 box pack of 100 Fisher 14-829-10D 15.94  
Pasteur transfer pipets 1 box of 400 Fisher 13-711-37 17.04  
immersion oil 0 case of 12 Fisher 12-369A 38.09  
PCR purification kits 125Rx 50Rx ClonTech 635960    
PCR Master Mix 60Rx 100Rx Fisher PRM7502 56.34  
parafilm 1 each Fisher 13-374-12 16.76  
trash beakers (tricorner 1000ml) 0 case of 100 Fisher 02-593-50F 51.22  
aluminum foil 1 roll grocery store      
plastic spoons 1 box grocery store      
Petrov-Hauser counters 12 each Fisher 02-671-54 101.07  
hockey sticks 24         handmade from 1/8" glass rod 
EtOH bottles w/ EtOH for flaming 0   Stores      
gas generators for gaspacks 4 10-pack Fisher B71040 12.15  
Kimwipes 0 60/box Fisher 06-666A    
gloves all sizes box of 100 Fisher 19-041-190C 9.81  
safety glasses 60 each Fisher 19-041-712 1.35  
wire racks for Wino columns 12 each Fisher 05-663-103  12.74  
4" box fans for incubators 0 each RadioShack - ~20.00  
Chemicals            
peptone 250g 500g bottle Fisher DF0118-17-0    
yeast extract 200g 500g bottle Fisher DF0886-17-0    
dextrose 200g 500g bottle Fisher DF0155-17-4 14.45  
NH4SO4 25g 1kg b ottlee Fisher BP212R-1    
K2HPO4 25g 500g bottle Fisher BP363-500    
MgSO4-7H2O 25g 500g bottle Fisher M65-500    
NaHCO3 250g 1Kg bottle Fisher BP328-1    
Na2HPO4            
KH2PO4            
CaCl2-2H2O mg          
NaCl 100g 3.5Kg bottle Fisher BP358-212    
CaSO4 500g 500g bottle Fisher AC31525-5000 45.4  
CaCO2 (chips) 500g 2Kg bottle Fisher AC403790020    
glycerol 50ml 100ml Fisher DF0282-15-2    
Instant Ocean     FishPros     Use from aquarium
ampicillin 2g 5g bottle Fisher BP1760-5 28.39  
agar 1Kg 500g bottles Fisher BP1423-500 88.44   
100ug/ml 515FPL     ICN      
100ug/ml 1492RPL     ICN      
mineral oil 10ml liter Fisher BP26291    
50X TAE 1 500ml bottle Fisher BP1332-500 29.83  
agarose 10g 100g bottle Fisher BP1356-100 148.41  
EtBr stain trace 10ml bottlee Fisher BP1302-10 27.8  
cellulose powder 100g 250g bottles Fisher ICN19149983 30  
EtOH 100ml   NCSU Stores      
100Kb MW ladder 20 lanes 50ug tube Fisher BP25551-50 144.8  
premixed LB for 10L 500g bottle Fisher BP1426-500 33.85