Revision as of 13:00, 12 August 2021

List of things to check regularly in 431

• Sterile water: Always at least one full bottle of autoclaved water
• 75% alcohol spray: Always at least one full spray flask of 75% alcohol
• DMEM ++: Always one half full bottle of DMEM supplied with extras
• Yellow waste container: no more than 3/4 filled
• sticky mat: Check that sticky mat sticks
• Vacusafe: Empty when it is more than half full
• Water in incubator: Once a week, refill sterile water in incubator. Need: 1-2 l.
• Diluted PBS: When the diluted PBS bottle is less than half full prepare another
• List: Check the list of things to buy (on the wall in the fore-room)
• CO2: Check the CO2 level in bottles. Reorder bottle if necessary.
• Pasteur pipettes: Always at least 20 sterile pasteur pipettes in hood, if not: autoclave new
• Coats: Every 3 weeks: wash coats
• Cleaning: Every Friday clean lab benches and hood.

List of things to check regularly in 420

So far the activities here do not require constant resupplies

List of procedures

Autoclaved water

Must be done 4-5 hours before use of water (for cooling down)

• Get 5L bottles of water from chemistry (check quality resistivity > 1 Ohms) or use the stock in 420
• Fill 1L borosilicate bottle with water
• Put cap on, untighten it slightly
• Put autoclave scotch the lid and the bottle. One piece of scotch should cover both
• Put in autoclave and follow procedure on autoclave

Dulbeccos Modified Eagle's Media, DMEM adding extras

• It is best to prepare a few tubes with the required amount of FETAL BOVINE SERUM (FBS) and Penicilin Streptomycin (Pen-Strep), and store them in the freezer. Each time only unfreeze the small tubes, instead of the whole bottle. As for 500 ml DMEM, it requires 50 mL (or 10%) of FBS and 5 mL (or 1%) of Penicilin Streptomycin (Pen-Strep).
• Step 1: Before prepare for DMEM solution, take out 1 tube of FBS and 1 tube pen-strep from the freezer and place them in the fridge (1 day in advance, after the overnight is fine).
• Step 2: work inside the LAF bench and add 50 ml FBS and 5 ml Pen-strep to the 500ml DMEM.
• Tight the caps, and slightly shake the bottle to mix them.  Label the bottle with names and date, then store it in the fridge.

Stock solution preparation:

FETAL BOVINE SERUM (FBS)  

• Step 1:  Use 75% antibac ethanol alcohol to spray both hands with gloves, then the surface of the FBS bottle, the surface of 25 ml Serological Pipette, a few of 50 ml conical tube, and place them inside the LAF fume hood.  
• Step 2: Inside the LAF hood, completely loose the conical tube caps and placed them in frame holder. Transfer 51 ml FBS into each conical tube, and tight the caps.  
• Step3: label each conical tube, put them in a plastic bag, and store them in the freezer. 

  Penicilin Streptomycin (Pen-Strep)  

• Use the same procedure as FBS, and each conical tube with 5,5 ml Pen-strep, then store them in the freezer with labels. 

Vacusafe

• Step 1:  Add 50 ml chlorine (around this amount) to the bottle.
• Step 2: find one autoclave bag in v420, pour all into the bag and tight the bag.
• Step3:  put the waste bag into yellow container. 

Refilling water in incubator

• Put on gloves and spray gloves
• Have 1-2L sterile water (autoclaved type II, not type I) ready and spray the outside
• Open incubator
• Withdraw water pan half way
• Pour water in to half full (around 1.5L total)
• Once a month, get the tray out, empty it completely and clean it with ethanol before filling it back. While the tray is out, swipe remains of water at the bottom of the incubator and clean this part with ethanol.

Preparing diluted PBS

Instruction:

1^st: Transfer 50 ml PBS stock solution into the empty 500 ml borosilicate glass bottle (work inside the bio LAF bench to avoid polluting the stock PBS solution)

2^nd: Transfer 450 ml type II water into the same above 500 ml borosilicate glass bottle. (work outside the LAF bench, on the lab bench)

3^rd: Autoclave the diluted PBS solution before using.

Required Items:

25 ml Serological Pipette, 500 mL volume metric flask, empty 500 ml borosilicate glass bottle 

Stock solution:  (PBS - PHOSPHATE BUFFERED SALINE 10× concentrate - P5493) from sigma / Merck, Type II water

Work Steps

Step 1:  Use 75% antibac ethanol alcohol to spray the pack surface of 25 ml Serological Pipette, the surface of the 500 ml borosilicate glass bottle and the bottle of stock solution.

Step 2: right after spray each item and place them inside the bio LAF hood.

Step 3: Transfer 50 ml PBS stock solution into the empty 500 ml borosilicate glass bottle and close the cap, then take out the bottle from LAF.

Step 4: transfer 450 ml type II water with 500 mL volume metric flask into the 500 ml borosilicate glass bottle. (Work outside the LAF fume hood – just on lab bench).

Step 5: Autoclave the diluted PBS solution.

Autoclaving pasteur pipettes

• stock in 420 or in cupboard in 431
• put them in a 1L beaker, tips at the bottom, beware not to break any
• put some aluminum foil on top to close the beaker and fasten it into place with autoclave tape
• put it in the autoclave and follow the autoclave's procedures

Waste disposal

PDF Norwegian

place to store the Yellow waste bins.

UK Floor close to the waste containers place. At the entrance of the loading place, there is a wood door with lock. We need borrow the key from Runar Staveli runar.staveli@kjemi.uio.no from chemistry store in the chemistry building (return the key right away after storing the yellow waste bins).

Take several empty yellow bins at the same room to store the yellow waste store bin.