• General Rules
• Buffer Storage
• Cell Storage
• Glassware Protocols
  • For Flasks and Bottles
  • For Culture Tubes
  • Washing Glassware
  • Culture Tube Processing
• Microbiology Protocols
  • Making a Gel
  • Sterile Technique
  • General Media Tips
  • Pouring Agar Plates
  • Streaking Technique
  • Innoculating Single Colonies from Plates to Liquid Media
  • How to do a serial dilution
• E.coli protocols
  • How To Isolate DNA (crude total nucleic acid prep)
  • Making Chemically Competent E.coli Cells
  • Transformation Of Chemically Competent E.coli by Heat Shock
  • Transformation of Electrically competent cells by Electroporation
• Lab Equipment Protocols
  • Using the pH Meter
  • How to use Nanodrop spectrophotometer
  • Cell Counting Absorbance Measurements with the Ocean Optics Spectrometer
  • Cleaning Gel Dock Station
  • How to Autoclave
  • Using Hemocytometer
• Phage Protocols

General Rules

Always use gloves in the Gel area

Don't wear lab coat outside of lab or while eating

SAFETY FIRST

When handling acids and bases, wear gloves!

Centrifuge anything that comes out of the fridge or freezer!

ALWAYS DATE YYYY.MM.DD

Buffer Storage

• Liquid media can be kept 4-5 months @ 4° C.
• Sugar stocks, amino acid stocks, can be kept 6 months @ 4°C.

Cell Storage

• cells on Agar plates are stored 2-3 months if sealed in bag or parafilm
• cells in liquid are stored 2-3 months
• cells stored at -80 C are kept permanently (glycerol is added to prevent cells from lysing).

Glassware Protocols

For Flasks and Bottles

1. Rinse with tap water
2. All Labels removed by the user
3. Place glassware (with a little water in the bottom) in the dirty
4. glassware bins below the vacuum station## Place cap in the cap washing basket

For Culture Tubes

1. Empty any cell cultures into the Wescodyne killing container.
2. Place yellow caps into cap soaking container.
3. Submerge tubes into the Wescodyne Soaking beaker. TUBES SHOULD BE SUBMERGED NOT FLOATING! If there is not enough water – fill the beaker with tap water. The concentrated Wescodyne solution is below the sink.
4. Tubes must be loaded into the square washing basket in order to have them washed by the 8th floor facility. When the soaking beaker is full, they can be transferred to the basket for washing. PLEASE do not move them to the washing basket unless you are taking them to the glassware facility immediately – they will dry with contaminants at the bottom of them.

Washing Glassware

1. The glassware facility is Room 825. The director’s name is John.
2. Get cart from Room 1237.
3. Empty water from Flasks and Beakers and place into autoclavable tray (translucent – not white buckets)
4. Take glassware to John. There is an elevator key labeled “E” on the key rack if you wish.
5. DO NOT LEAVE THE GLASSWARE FOR DAYS. You should ask John when to return.
6. When you bring the glassware back to the lab, please wrap all bottle and flask necks with aluminum foil.
7. Bottle caps and magnets are wrapped separately.
8. Culture tubes should be placed upright in a blue rack with gloves, and the yellow caps placed on them. Please place the rack of covered tubes on the tube shelf where it says “Washed Only”
9. Place washed spatulas back into the hood area.

Culture Tube Processing

1. Take a rack of tubes from the “Washed only” section of the tube shelf.
2. Make sure caps are on all tubes.
3. Place a piece of aluminum foil over the entire rack of tubes. Mark the foil with a piece of autoclave tape.
4. Autoclave the tube rack (with the marked foil) on the instrument setting (looks like scissors on the button)
5. Place tube rack into 65 degree dying oven (gray) overnight to dry out the moisture from the autoclave cycle

Microbiology Protocols

Making a Gel

• Agarose concentration: 1%

* Obtain 1.0g of LE agarose
* Add 1.0g of agarose into ET Br flask
* Add 100ml of 1x TAE to flask
* Microwave flask for 1 min
* Take out flask from microwave (HOT!!)
* Place in microwave for another minute (make sure it's boiling)
* Take out of microwave and allow to cool
* Add 5ul of ethidium bromide to flask
* Swirl contents
* Pour contents into the agar plates
* Allow gel to harden

Sterile Technique

• Never put caps on the bench or hold them too much
• Flame the pipet (especially between dips)
• Flame a bottle top upon opening it.
• try to really minimize the time in which the cap is off the bottle

General Media Tips

500ml plate media = 1 sleeve petri plates.

• For "TUTT," however, we only fill 500ml bottles to 400ml.
• Tape/label bottles
• For community autoclaves place bottles in autoclave safe bucket. And there should be about 1-2 " water at the bottom.

Pouring Agar Plates

• Open a bag of empty plates, and save the wrapping.
• Stack empty plates, upside down, about 10 high.
• Its best to pour YPD Agar into plates while its hot.
• Using one hand to hold the bottle and the other to lift the stack, start pouring the YPD Agar into empty plates, starting from the bottom.
• After filling all plates, dont forget to label the plates, using the corresponding color of the media, labeled on the referigerator. A convinient way is to hold the stack down with one hand and use the other to vertically mark the plates from bottom up.
• Put the plates in incubator, again upside down overnight.
• Observe the growth next day.
• Now, to store them in refrigerator, put the poured plates carefully in the saved plastic bag. Make sure to close the bag with a potato chip fold(Prof. Goddard said it works the best). Close the bag with your favourite colored tape and label it.

Streaking Technique

• IF STREAKING CELLS FROM -80 FREEZER, ALLOW BLOT TO DRY BEFORE STREAKING
• pipet 5 microliters of liquid yeast/bacteria onto the side of an agar plate.
• allow blot to dry
• using a stick, gently (so as not to dig into to agar) streak first section. (See photo)
• switch to opposite side of stick. Begin streaking second section in the last line of the first section (so as to grab yeast or bacteria).
• for third section repeat process using the same side of the stick.
• stack and invert plates
• store plates overnight in temperature specific incubator. 30 degrees for yeast. 37 degrees for bacteria.

Innoculating Single Colonies from Plates to Liquid Media

• If your plates have been well struck, single colonies will be visible. Each colony arises from a single cell - hence they are clonal.
• Into an empty STERILE test tube, pipet 2 microliters of LB.
• If you need to double dip make sure to flame the serological pipet.
• Choose a single colony from the agar plate by touching the tip of sterile pipet tip to the colony.
• Release pipet tip into the test tube.
• Incubate test tubes in incubator shaker at 37° ^oC.
• make sure to have a control test tube with just the YB sln.

How to do a serial dilution

Note: In order to do a serial dilution and count the number of cells you will need to have innoculated a single cell colony and grown it over night

Materials
10 small tube capsules (not as small as PCR size)
For diluting sol. you can use our tris EDTA buffer or LB for e.coli or YPD for yeast.

E.coli protocols

How To Isolate DNA (crude total nucleic acid prep)

1. Pipet 600 μL of cells from an overnight culture tube in a clean 1.5mL snap cap tube.
2. Centrifuge tubes at 5000 RPM for 5 minutes. Make sure hinges are facing the outwards.
3. Remove supernatant and discard it.
4. Resuspend cell pellet in 200 μL of buffer # 1 (Tris HCL and EDTA). Pipet up and down or vortex to resuspend cells. Make sure there are no clumps. Also don't over vortex!
5. Lyse the cells with 400μL buffer #2 NaOH/SDS. pipet 400µL. Invert tube gently to mix. Do NOT vortex. The solution should be clear.
6. Add 400µL of 3M KAc pH5(Buffer 3)This separate proteins from the nucleic acids. Proteins will coagulate and denature. Invert gently to mix. Wait ~5 min
7. Centrifuge for 20 minutes @ 12,000 RPMs.
8. Save supernatant (this is the DNA!)
9. Add 700µL of 100% Isopropanol
10. Store in -80 ° for 30 minutes.
11. Centrifuge for 25 min @ 12,000 RPM. A pellet should be visible at the tip of the tube. IF YOU SEE PELLETS, discard supernatant.
12. To reduce salt concentration in pellet, pipet 100µL of 70% Ethanol into the tube and loosen the pellet from the wall.
13. Centrifuge for 10 min @ 12,000 RPM.
14. Pipet off ethanol (careful of the pellet) and invert tubes to dry overnight

Making Chemically Competent E.coli Cells

1. Inoculate an overnight starter culture (appropriate media) with a single colony or from frozen culture, grow at 37 ° C
2. The next day, inoculate 50 mL appropriate media (LB +/- selection) in a 250mL shaker flask and incubate at 37 ° C in a rotary shaker at ~150 rpm, until OD600= 0.2-0.6 (early to mid log).
   • OD can be checked in nanodrop or bioscreen
3. Fill Ice bucket on the 8th (dishwashing room),9th (939HN),13th, or 14th floor
4. Decant the 50mL into conical 50mL falcon tubes. Bury into ice for ten minutes.
5. Balance the tubes in the Hettich centrifuge (opposite sides - same volume in each tube) and spin low speed (2500g) for ten minutes at 4 ° C.
6. Discard supernatant and resuspend cell pellet by pipetting up and down with large pipet tip, in 20 mL of chilled "Magic" Transformation buffer (MTB).
   • MTB is usually stored in the 4 ° C fridge.
7. Place back into ice for 10 minutes.
8. Spin again at the same speed for 10 min. While spinning, add .28 mL DMSO to 3.72 ice cold MTB.
9. Discard supernatant.
10. Resuspend in 2 mL of MTB+DMSO.
11. Place back into ice and Aliquot in 200uL batches into the -20 degree ice chest with aluminum insert. (or freeze directly in liquid nitrogen/Ethanol bath).
    • color of tubes should be noted on index card in the competant cell storage box
12. Store at -80 ° C . BE CAREFUL TO NEVER LET THE CELLS HEAT UP!!!!

Transformation Of Chemically Competent E.coli by Heat Shock

1. Fill Ice bucket in 825N (washing room),9th (939HN),13th, or 14th floor
2. Label Falcon 2059 snap cap tubes with cell line + plasmid names and bury in ice to chill
3. Take appropriate competant cells from -80 freezer and thaw on ice
   • aliquots are either 100uL or 200uL - 100uL per reaction is common
4. Thaw cells on ice/on top of ice until they are just melted. Then pipet 100uL into each appropriate falcon 2059 chilled tube.
5. Pipet 5-10uL of plasmid stock (depending on concentration) directly into the cells and keep on ice for 15-30 minutes.
6. Check water bath! - should be at 42^oC
7. Prepare "Faux" SOC media. 9mL LB, 1mL 40% glucose stock solution, 100uL 1M MgCl₂, 100uL 1M MgSO₄ - place in 37^oC incubator to bring to temperature.
8. Carry the ice bucket over to the water bath. Using a timer, submerge the bottom of the chilled tubes into the water bath (do not shake/aggitate) for 30s. You can to 2-4 at a time if they are well separated.
9. Remove tubes from water bath and bury back intop the ice for 2-5 min.
10. Add .8mL of Faux SOC medium and incubate in Innova Shaker at 37^o for 1 hour.
11. Remove appropriate plates with selection marker from 4^oC refrigerator and open to check for excessive condensation. Allow plates to come to room temperature. Leave them inverted and open to dry if they need to.
12. Check for Spreaders - if none exist - then make some.
13. Remove tubes from Innova and label plates.
14. Pipet 200uL of solution onto each corresponding plate. Flame spreader and then place on an area of the plate that does not touch the cells to cool. Spread liquid over the entire plate. Using a lazy susan will help.
15. Allow liquid to dry on the surface of the plate. It's ok to leave the lids slightly ajar during this step. Invert and incubate overnight at 37^oC (unless you have plasmids that need 30^oC)

Transformation of Electrically competent cells by Electroporation

1. Pick some colonies from desired strain and grow in culture tube containing 2mL of LB and required antibiotic inside shaker until OD₆₀₀ is around 0.4.
2. Aliquot 1mL of culture into two 1.5 mL centrifufge tubes.
3. Chills cells in ice-water bath for 10 minutes.
4. Centrifuge the cells for 10 minutes at 4^oC at 8000rpm.
5. Pipette off supernatant and discard. Resuspend pellets in mL ice-cold distilled water.
6. Centrifuge cells for another 10 minutes at 4^oC at 8000rpm.
7. Resuspend pellet in 50uL of ice-cold distilled water.
8. For electroporation step, include two conditions: +PCR (with PCR fragment) and -PCR (no PCR fragment. this is your control).
9. Chill electroporation cuvettes for 5 minutes on ice.
10. Add 5pg to 0.5ug of your PCR amplified-DNA to cells.
11. On the Bio-Rad? gene Pulser X-cell, use the pre-set values for your cell-type (bacterial, mammalian, fungal).
12. Take electroporation cuvettes out of ice and wipe off any excess water from the outside of the cuvette.
13. Place cuvette into sample chamber and apply pulse by pushing red button.
14. Remove cuvette and immediately add 1mL of LB medium. Transfer to a sterile culture tube.
15. Incubate for 60-120 minutes with moderate shaking at 37^oC.
16. Place aliquots of the transformation culture on LB plates supplemented with required anitibiotic and incubate overnight at 37^oC.

Lab Equipment Protocols

Using the pH Meter

• Turn machine On
• If machine is calibrated, you will see the words AUTOLOCK, STAND and SLOPE across the top of the machine.
• If not, press mode, place pH meter tip in the pH 7 solution. Wait for machine to read 7 (or close to it) and stop flashing the word WAIT. It will say HOLD. Once WAIT disappears, remove pH meter tip and rinse with dionized water. Now submerge the pH meter tip in the pink acid or blue base (depending on what you will be measuring). And press slope. Wait for machine to stop flashing and for the word HOLD to come up.

Now the machine is calibrated!

• Rinse pH meter tip. Submerge tip in solution you wish to pH. Now press measure.
• You should not have to recalibrate between sequential usages, but if you do, you know what to do!

Always store the pH electrode in Pink(PH=4)buffer. The constant use of the PH Buffer in strong or weak solutions can reduce the lifetime of the electrode by slowly dissolving the glass membrane.

How to use Nanodrop spectrophotometer

• Wipe machine base and cap attached to fiber optic cord with soft tissue(kimwipes)
• Open program called Nanodrop on the desktop
• It will provide you with a list of the nature of the samples you will be measuring
• Load a sample of water to initialize the machine
• Use another sample of water or in most cases a buffer to take a blank measurement by hitting the blank icon to get measurement to zero
• Between samples wipe machine with kim wipe
• When loading samples put the number of the sample in the order of how its run in the box "sample ID"
• click on measure
• Follow the above procedures for every sample but you do not have to blank after the first continue to wipe machine, load the sample, indicate the order of the sample by writng for example 5 in the ID box, then hit measure
• When you are finished place a kim wipe in the nanodrop to indicate that it is clean.
  • IF YOU ARE COUNTING NUMBER OF CELLS, CLEAN NANO-DROP after using with 70% EtOH.

Remember if you are taking samples from the refrigerator, habitually centrifuge!

Cell Counting Absorbance Measurements with the Ocean Optics Spectrometer

1) Make sure the spectrometer is configured for absorbance measurements (fibers should be 180 degrees to one another.
2) Turn on blue lamp and allow to warm up for a few minutes.
3) Turn on Jaz Spectrometer (red LED will light) and open Spectra Suite Software.
4) Choose from menu "New"-> "Absorbance Measurement"
a. A window will appear and the Jaz unit should be visible (click Next)
b. Make sure the shuttter is open on the blue lamp. The spectra of the lamp should be shown on the screen. The peak should be flat(or saturated) in the middle and have a shoulder peak at 40,000. If not, increase the amount of light by turning the brass knob on the front of the lamp.
c. Choose acquisition rate(100ms)
d. Click Next
5) The window should display a yellow light bulb. Place your reference sample(ex.LB) in the cuvette holder and cover the lid. Click on the yellow light bulb to store the reference spectra. Click Next
6) The window should display a dark light bulb. Close the shutter on the blue lamp. The spectra should drop to a flat line in the widown. Click on the dark bulb to store the dark spectra. Click Next
7) The instrument is now ready to measure samples.-------
8) Insert first sample and choose the wavelength(at bottom of spectra window) which you would like a vlaue for. Common wavelengths for cell densities(420nm and 600nm). Reopen shutter.Click pause to get a stable reading.Record the number in your notebook or spreadsheet.

Notes***
1) The lamp heats up over time. You will need to update your reference spectra if you leave the lamp on for more than 1hr.
2) PLEASE turn the lamp off after use.

Cleaning Gel Dock Station

• Gloves on!
• Remove gel and place into container in gel area to dry
• Don't be afraid to touch your gel.... In fact, Dr. Spangler encourages it! Just make sure your gloves are on!
• Take water bottle from gel station and spray water onto surface
• Take paper towel and wipe dry
• Remove gloves
• Take new paper towel and place on top of surface
• This indicates that the area has been wiped down
• Place camera back onto dock
• Remove paper towel before re-using
• Repeat for each use

How to Autoclave

1. make sure the caps are loose on the bottles you put in the autoclave and they are taped with autoclave tape.
2. make sure there is enough water in the machine.
3. choose setting: bottles, utensils, clothes?, and the fourth possible setting.
4. make sure time and temp are correct
5. make sure device is closed tightly.
6. run
7. when opening autoclave where gloves and stand away from opening of machine to avoid steam
8. note that gloves are not waterproof and that getting them wet will severely burn you.

Using Hemocytometer

- Add a few drops of cell suspension to chamber of the hemocytometer
- Place the cover slip and the suspension will fill the chamber through capillary action
- Observe under the microscope anywhere between 40x and 100x or until individual cells are observable
- Count individual cells in the corner squares labeled A that include 16 squares within them
- Average all 4 A corners together and multiply by 10^4

Your total cell count is that number/mL

Phage Protocols