Protocols

Protocols kdorfman Tue, 06/16/2009 - 20:02

How to do stuff

How to get containers and stickers from EH&S

Sterilization

Sterilization kdorfman Tue, 06/16/2009 - 17:50

Sterilize anything that cells might grow in.

Autoclave glassware, tips, and microfuge tubes.

Autoclave most media and salt solutions.

Do not sterilize by autoclave items containing:

  • detergents (e.g., SDS) - they can boil over

  • heat sensitive ingredients (e.g., vitamins, hormones, antibiotics, proteins)

  • sugar in growth medium (the sugars and amino acids may react together, reducing the concentration of both)

  • HEPES

  • DTT (dithiothreitol)

  • Beta mercaptoethanol

  • corrosives (e.g. acids, bases, phenol)

  • solvents or volatiles (e.g. ethanol, methanol, chloroform, acetone, formaldehyde, formalin or glutaraldehyde)

  • chlorine (e.g., bleach)

  • anything radioactive

Filter sterilize any liquid that must be sterile, and that you cannot autoclave.

biohazard_waste_only.docx

Autoclave

Autoclave kdorfman Tue, 06/16/2009 - 20:25

autoclave manual

Always put a piece of autoclave tape on the item or container so you can tell if it was exposed to the steam.

If the green light is on, press the red reset button.

  • Biohazard Waste: See here: https://wahoo.nsm.umass.edu/content/biohazard-waste

  • Tips: Load racks into boxes wearing gloves (this caution is primarily for RNA work, as most people's skin has RNase on it).
    15 minute sterilization; 40 min drying time; open autoclave CAUTION - HOT! to let steam escape.
    If there is too much condensation inside the boxes, put them in the oven at ~60C for ~an hour.

  • Microtubes: Put into 600 mL plastic jars, screw cover on loosely, so the steam will penetrate. Same time as for tips.

  • Liquids: Larger volumes require longer sterilizing times. Use this table:

Largest volume (mL) Minimum time (min)
75 25
250 30
500 40
1000 45
1500 50
2000 55
>2000 55 + 10 per L
  • Monthly spore test EH&S recommends Fisher 12-001-1 population 10^5 Prospore Bacillus stearothermophilus
To sterilize mL of liquid medium
autoclave for: minutes

Minutes = 8.3853 * volume^0.2449

autoclave_log.docx
autoclavemanual.pdf

Monthly Maintenance

Monthly Maintenance kdorfman Fri, 09/09/2016 - 15:45

  • Turn generator switch off

  • Let cool to ~5 lb pressure

  • Turn master switch off

  • Open valve

  • Let tank drain

  • Turn generator switch on

  • Close valve

  • Let it fill

  • Turn master switch on

Autoclave bags

Autoclave bags bcrcstaff Thu, 02/14/2019 - 13:45
Container dimensions (in) bag size
Rubbermaid 16" x 10" 24" x 36" Fisher 01-8143
Round diam = 18" 38" x 48" VWR 14220-044
Square 16" x 14" 38" x 48" VWR 14220-044

Dishwashing

Dishwashing kdorfman Tue, 06/30/2009 - 20:35

There are dishwashers in rooms 261 and 361.

Instructions are in the drawer labeled "manuals" in each room, and linked to this page. "Dishwashing" gives general user instructions; "Dishwash-program-guide" explains how to change the cycles - for advanced users only.

Replacement detergent: Fisher 04-319B Thermo Scientific* Nalgene* L900 Liquid Detergent

1 gal $59

4 gal $167 (@$42)

Dishwashing.pdf
Dishwash-program-guide.pdf

Drierite Regeneration

Drierite Regeneration margaret Thu, 10/06/2011 - 18:34

For crystals: 1 hour at 210° C (=425° F) in shallow glass pan. See details below:

For cartridge: 3 hours at 150° C (=~300° F), perforated top down. Blue means full reactivation.

REGENERATION OF DRIERITE DESICCANTS After normal use, any of the forms of DRIERITE may be regenerated for reuse. The operation is simple and involves only standard equipment. The used and exhausted desiccant should be ventilated to remove vapors, if any, and stored in a convenient container until a sufficient amount is accumulated to justify the work of regeneration.

Regular and Indicating DRIERITE For the regeneration of Indicating DRIERITE and small lots of Regular DRIERITE , the granules may be spread in layers one granule deep and heated for 1 hour at 210° C or 425° F. The regenerated material should be placed in the the original glass or metal container and sealed while hot. The color of the Indicating DRIERITE may become less distinct on successive regenerations due to the migration of the indicator into the interior of the granule and sublimation of the indicator.

The Importance of Temperature The temperature at which DRIERITE desiccants are regenerated is crucial in restoring DRIERITE to its original condition. Absorbed moisture is water of hydration and is chemically bound to the calcium sulfate of DRIERITE. Temperatures in the range of 400° - 450° F are required to break these bonds and release absorbed moisture. Lower temperatures, regardless of heating time, will not regenerate DRIERITE unless applied under vacuum (28" Hg, 325° F or 26" Hg, 275° F). Care should be taken not to overheat DRIERITE Desiccants. High temperatures can alter the crystal structure and render the desiccants permanently inactive.

Mini-prep

Mini-prep kdorfman Thu, 09/15/2022 - 15:33

Keep stocks of 2 mini-prep kits:

Qiagen

Zymo Classic D4015

Qiagen

Qiagen kdorfman Thu, 09/15/2022 - 19:53

Jeff's CMBL miniprep

Zymo miniprep classic

Zymo miniprep classic kdorfman Thu, 09/15/2022 - 19:53

For many labs

Before beginning, add 95% ethanol to Plasmid Wash Buffer 4:1 Also, once RNAse has been added to P3 (yellow), keep it at 4C.

Protocol

  • Spin 0.5 - 5 mL bacterial culture in 1.5 mL tube 20 sec full speed

  • Add 200 uL P1 (red), resuspend pellet

  • Add 200 uL P2 (green); mix by inverting 2-4 times. Clear, viscous, purple solution indicates cell lysis

  • Add 350 uL P3 (yellow). Mix thoroughly. DO NOT VORTEX. Turns yellow when neutralization is complete.

  • Incubate RT 1-2 min

  • Spin 2 min

  • Put column into collection tube, transfer supernatant. DO NOT DISTURB GREEN PELLET (I found it wasn't green, but OK)

  • Spin 30 sec

  • Discard flow through, put column back into emptied collection tube

  • Add 200 uL Endo-Wash Buffer to the column, spin 30 sec

  • Add 400 uL Plasmid wash buffer to the column, centrifuge 1 min

  • Transfer column to clean 1.5 mL microcentrifuge tube

  • Add 30 uL DNA Elution Buffer

  • Spin 30 seconds. Plasmid DNA is in the collection tube

Students need per reaction1

  • 1 mL (?) overnight culture of transformed cells (or spun down pellet of ON culture)

  • 1 column, 1 2-mL collection tube

  • sterile 1.5 mL tube

  • ~200 uL P1 (red)

  • ~200 uL P2 (green)

  • ~350 uL P3 (yellow)

  • ~200 uL Endo-Wash-Buffer

  • ~400 Plasmid Wash Buffer

  • ~30 uL DNA Elution Buffer

  1. All the ingredients are sold separately, so it's OK to give students some extra to allow for pipetting errors. ↩︎

ZR Plasmid Miniprep Classic

Peroxide testing

Peroxide testing kdorfman Tue, 10/09/2018 - 20:46

Preparation

  • Samples containing more than 25 mg/L H2O2 (Cat. Nos. 110011) or 100 mg/Ll H2O2 (Cat. No.110081) must be diluted with distilled water or peroxide-free ether.

  • The pH of the aqueous sample must be within the range 2-12 If necessary, buffer the sample wit sodium acetate or, respectively, adjust the pH with hydrochloric acid

Test Procedure

  • For aqueous solutions:

    • Immerse the reaction zone of the test strip in the pretreated sample (15 – 30 C) for 1 sec.
    • Allow excess liquid to run off via the long edge of the strip onto an absorbent paper towel and after 15 sec (Cat. No. 110011) or after 5 sec (Cat. No. 110081) determine with which color field on the label the color of the reaction zone coincides most exactly.
    • Read off the corresponding result in mg/L H2O2

  • For organic solvents:

    • Immerse the reaction zone of the test strip in the pretreated sample (15 – 30 oC) for 1 sec.

    • After the solvent has evaporated (gently fan the strip back and forth for 3 – 30 sec), immerse the reaction zone in distilled water for 1 sec and allow excess liquid to run off via the long edge of the strip onto an absorbent paper towel.

    • After 15 sec (Cat. No. 110011) or after 5 sec (Cat. No. 110081) determine with which color field on the label the color of the reaction zone coincides most exactly.

    • Read off the corresponding result in mg/L H2O2

General Notes

  • any blue within 3 minutes is a positive result

  • If the color of the reaction zone is equal to or more intense than the darkest color on the scale or in another color emerges, repeat the measurement using fresh samples diluted with distilled water or, respectively, peroxide-free ether until a value of less than 25 mg/L/ H2O2 (Cat. No. 110011) or 100 mg/L (H2O2) (Cat. No. 110081) is obtained.

In the case of Cat. No. 110081 the reaction zone indicates values within the measuring range also for H2O2 contents from 5000 mg/L (0.5 %) up.

peroxide_forming_material_handout2018.pdf

Weekly Inspections

Weekly Inspections kdorfman Tue, 10/11/2011 - 16:47

Biohazard Waste

Biohazard Waste kdorfman Tue, 10/11/2011 - 16:50

Check the red biohazard trashcans, usually found in 360, 364, or 368.

If it is more than half full, or if it really stinks, autoclave it.

Wear gloves! Put the clear bag into a Nalgene autoclave basket, but don't seal it up tight. (If you do, it will explode in the autoclave.) Double bag it if it has a lot of liquid in it.

Set it for 60 minutes sterilization, liquid cycle.

When it is done, make sure the "autoclaved" sign on the bag has turned dark, then seal the bag and put a non-hazardous waste sticker on it, put it inside a black trashbag, and throw in the regular trash.