Edited: January, 2014
All lab members are expected to do the following lab duties
Maintain individual bench areas.
Help keep common areas (sinks, bacteria station, and gel bench) clean each day.
Receive and unpack boxes as they arrive.
Order items in advance of depleting the last of the item. Most items should always have one extra. For example, we stock 1 extra box of each type of tissue culture plate, 1 extra box of nitrocellulose, 1 extra box of whatman papter etc… Fill out order form or write item and catalogue number on board.
Maintain the tissue culture incubator that you are using.
Dispose of radiation waste as it is generated and monitor lab areas for radiation. If you are using radiation you will be expected to know how to safely dispose of it and keep everyone else in the lab safe as well.
Tie up the biohazard bags in the tissue culture room and the bacterial biohazard waste.
The flasks in the tissue culture room need to be emptied and cleaned every day. Add bleach to the flask before pouring down drain, rinse out flask and clean, then add approximately 10 ml of bleach to bottom of flask before replacing. Please do this after you are finished with your TC for the day.
Monitor the reagents/plates that you will need for your experiments and alert the appropriate person in advance if you will be using an unusually large amount of a common item.
Lab chores for Dave
Maintaining CO2 and Liquid Nitrogen
Nancy Lab Duties
Dishwashing/Autoclaving
Media Preparation
Maintain at least 40 LB-Amp plates, 40 LB-Kan plates, 10 LB plates, 2L of LB in various aliquots, 100 ml of SOC media in small aliquots, and 2L of autoclaved water. Also make aliquots of 1000x stocks of ampicillin and kanamycin. Check the levels of these items on Monday and Wednesday of every week.
Tissue Culture Maintenance
Water bath cleaning – the tissue culture and lab water baths should be emptied, washed with ethanol, and refilled with deionized water plus 4 drops of algaecide (water bottle in tissue culture cabinent) once per week.Maintain supply of autoclaved Pasteur pipettes for tissue culture. Refill canisters, autoclave, stock in cabinet.
Sending and receiving reagents
Handle all reagent requests. Send reagents and keep record of all reagents shipped. Also when we receive reagents from other labs please make the appropriate stocks.
Gloria Lab Duties
Hazardous chemicals
Ensure the laboratory is in compliance with regulations concerning labeling of wastes, posting of signs, etc.
Ordering
Take a weakly inventory of commonly ordered supplies and order as appropriate. Order supplies listed on the board as needed. Maintain files of purchase orders. Maintain record of received orders. Follow-up on any ordering problems. Solicit quotations for equipment and expensive items.
Equipment maintenance
Coordinate repairs of equipment as needed.
Freezer defrosting
Defrost freezers as necessary.
Lisa Poole
50X TAE — Maintain > 250 ml at all times
10X Running Buffer –- Maintain >1L at all times
Cory Holland
Scrape frost from -80 freezers (at least once per month).
Replinish water in the TC incubators
Huzefa Dungrawala
2X Sample Buffer — maintain >20 250 ul aliquots at all times
6X Sample Buffer – maintain >10 50ul aliquiots at all times
1M DTT — maintain >10 100ul aliquots at all times
Gina Kavanaugh
FBS – aliquot for tissue culture as needed
Akosua Badu-Nkansah
5mg/ml aprotinin – maintain >10 20ul aliquots at all times
5mg/ml leupeptin — maintain >10 20ul aliquots at all times
100mM sodium vanadate — maintain >10 100 ul aliquots at all times
1M B-glycerolphosphate – maintain >10 500ul aliquots at all times
Clint Caroll and Kareem Mohni
10X TBST – maintain >1L at all times
Jessica Luzwick
4X Upper Gel Buffer — Maintain >250 ml at all times
4X Lower Gel Buffer — Maintain >250 ml at all times
Move supplies into tissue culture as needed (check twice per week), check and fill water in CO2 incubators as needed. Dispose of glass pipette waste in tissue culture room. Tape up box so no glass can escape. Write “glass waste” on top and place in hall.
Kami Bhat
Radiation waste disposal: Ensure proper labeling of waste containers (needs to be done when containers are started). Request radioactive waste pick-up.
Instructions for reagent preparation:
50 X TAE
57.1 ml Glacial acetic acid
100 mls 0.5 M EDTA or 250 mls 0.2 M EDTA
242 g Tris
Total volume 1L – Make Solutions using deionized water
10 X TBST
24.2 g Tris
80 g NaCl
10 mls Tween
Add above chemicals to 850 mls of deionized water
Adjust pH to 7.5 with HCl
Bring volume to 1L
Filter sterilize
10X Running buffer
30.2 g Tris
144 g Glycine
10g sodium dodecyl sulfate
Add above chemicals to 850 ml deionized water
Bring total volume to 1L with deionized water
4X Lower Gel Stock
90.86g Tris
2 g sodium dodecyl sulfate
Add above chemicals to 450 ml with deionized water
Adjust pH to 8.8 with HCl
Bring total volume to 500 ml using deionized water
Filter sterilize
4X Upper Gel Stock
30.3 g Tris
2 g sodium dodecyl sulfate
Add above chemicals to 450 ml with deionized water
Adjust pH to 6.8 with HCl
Bring total volume to 500 ml using deionized water
Filter sterilize
10% APS
1g Ammonium persulfate
10 ml milliQ water
Mix APS in water
Store at 4˚C
1000X AMP stock
1g Ampicilllin (4˚C)
10 ml autoclaved deionized water
sterile filter solution. Aliquot into 1.7 ml tube. Store at -20˚C
1000X KAN stock
0.5 g Kanamcyin (located at 4˚C)
10 ml autoclaved deionized water
sterile filter solution. Aliquot into 1.7 ml tube. Store at -20˚C
LB/LBA
| Volume | 1L | 2L | 3L | 4L | 5L |
| Tryptone | 10 g | 20 g | 30 g | 40 g | 50 g |
| Yeast Extract | 5 g | 10 g | 15 g | 20 g | 25 g |
| NaCl | 10 g | 20 g | 30 g | 40 g | 50 g |
use deionized water
LBA – Add 7.5 g agar per 500 ml bottle
Autoclave solution 45 min ; Liquid cycle
2X SDS Loading buffer
0.4 g sodium dodecyl sulfate
2 ml Glycerol or 4 mls 50% Glycerol
1.25 ml 1M Tris pH 6.8
0.01 g Bromophenol blue
bring total volume to 8 mls using milliQ water
Sterile filter
Aliquot into 1.7 ml tubes and store -20˚C
Add 1/5th volume of 1M DTT just prior to use. (ie 100 ul of 1M DTT per 400ul of 2X SB)
6X SDS Loading buffer
7 ml 4X Tris Cl /SDS (see below)
3.0 ml glycerol (30%final)
1 g sodium dodecyl sulfate
0.93 g DTT (0.6 M final)
1.2 mg bromophenol blue (0.012% final)
add dd H20 to 10 ml
store at – 70˚C
4X Tris Cl /SDS
6.05 g Tris in 40 ml milliQ water
adjust pH to 6.8
adjust to final volume of 100 ml
filter sterilize
add 0.4 g sodium dodecyl sulfate
store at 2-4˚C for up to 2 weeks
100 bp marker
for 250 ul
25ul DNA ladder (500ug/ml)
41.7 ul 6X loading buffer
183.3 ul milliQ water
store at room temp
200X Tryptophan
1.523 g Tryptophan
100 ml milliQ water
sterile filter
Store at -20˚C
Terrific broth
Solution 1
12 g Bacto Tryptone
24 g Yeast extract
4 ml glycerol (100%)
bring total volume to 900 mls using deionized water
Solution 2
2.31 g Potassium phosphate monobasic
12.54 Potassium phosphate dibasic
bring total volume to 100 mls using deionized water
Autoclave solution 1 and 2 and mix together afterward autoclaving
SOC (200 ml)
| Ingredients | Amount for 200 ml | Final Concentration |
| tryptone | 4 g | 2% |
| yeast extract | 1 g | 0.5% |
| NaCl | 400 ul of 5M | 10 mM |
| KCl | 500 ul of 1M | 2.5 mM |
| MgCl2 | 2 ml of 1M | 10 mM |
| MgSO4 | 2 ml of 1M | 10 mM |
| Glucose | 8 ml of 50% | 20 mM |
Combine ingredient and autoclave
Stock solutions:
for 1M KCl add 14.91 g / 200 ml milliQ water
for 1M MgCl2 add 39.58 g / 200 ml milliQ water
for 1M MgSO4 add 49.3 g / 200 ml milliQ water
Sterile filter each solution
Yeast minimal dropout Media (1L)
20 g Glucose
2 g dropout mix (see below)
900 ml water preheated in microwave for ~2 min
for plates add:
100 ul 5N NaOH
20 g agar
After autoclaving add 100 ml 10X YNB
if required, add 5 ml 200 X tryptophan (some trp is in the dropout mix, but if trp is required this should be included)
Dropout mix (see recipe sheet hanging on chemical hood adjacent to dry chemicals )
Yeast YPD media (1L)
10 g Yeast extract
20 g bacto peptone
20 g Glucose
add 1L water and autoclave
for plates add 20 g Bacto agar
10X YNB
50 g Ammonium Sulfate
17 g yeast nitrogen base
add water to 1L
filter sterilize and wrap in foil
store in fridge
Activation of Sodium orthovanadate
- Prepare 200 mM sodium orthovanadate
- Adjust pH 10.0 using either NaOH or HCl. (pH of sodium orthovanadate can vary by batch) At pH 10.0 solution will be yellow
- Boil solution until it turns colorless (approx 10 min)
- Cool to room temp
- Readjust pH to 10.0 and repeat step 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0.
- Store activated sodium orthovanadate as aliquots at -20˚C
( this procedure depolymerizes sodium orthovanadate converting it to a more potent inhibitor of protein tyrosine phosphatases)
Aprotinin (5mg/ml)
Use the total amount in the glass vial
If the total amount is 5 mg, then add 1 ml autoclaved milliQ water directly into the glass vial
If the total amount is 10 mg, then add 2 ml autoclaved milliQ water directly into the glass vial
Mix and aliqout 50 ul into 1.7 ml tubes labeled “A”
store in the freezer.
Leupeptin (5mg/ml)
Use the total amount in the glass vial.
If the total amount is 5 mg, then add 1 ml autoclaved milliQ water directly into the glass vial
If the total amount is 10 mg, then add 2 ml autoclaved milliQ water directly into the glass vial
Mix and aliqout 50 ul into 1.7 ml tubes labeled “L”
store in the freezer.
1M DTT
add 3.85 g Dithioerythritol (MW = 154.2) to 25 ml of milliQ water.
aliqout 500 ul into 1.7 ml tubes
store in the freezer.
6X DNA Loading Dye
0.05% bromophenol blue
0.05% Xylene Cyanol FF
30% glycerol in water