Helpful Protocols & Reference Materials

This page includes a list of DIY protocols used by SoundBio Lab. Some of these items were used in our workshops, others are used regularly by staff and members. 


Media Recipes

LB - Standard for most bacteria

  • 10g Peptone
  • 10g NaCl (or regular table salt)
  • 5g yeast extract
  • 15g agar (optional)

1 L distilled water

YPD - Great choice for growing yeast

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

 Adjust to pH 5 w/ dilute HCl

For TUBES -  dispense 10ml into each of about 100 16x150mm tubes, cap, & autoclave. Allow to cool, and add 40ul 25mg/ml ampicillin to each tube.

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.

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Antibiotic Stock Solutions

Ampicillin

100 mg/mL in H2O

To make 50 mL, add:

  • Quantity - 5g
  • Chemical- Ampicillin sodium salt (FW = 371.39 g/mol)
  • q.s. to 50 mL with water
  1. Filter sterilize, and aliquot 1 mL into sterile microcentrifuge tubes and store at -20°C.
  2. Use ampicillin at a minimum of 50 μg/mL in E. coli cultures. We often use ampicillin at 100 μg/mL. 150 μg/mL is the maximum that E. coli can generally handle.
  3. The presence of ampicillin in media is indicated by a single black line.
  4. Ampicillin is abbreviated as "AMP".

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Carbenicillin

50 mg/mL in H2O

To make 50 mL, add:

  • Quantity - 2.5g
  • Chemical - Carbenicillin disodium salt (FW = 422.36 g/mol)
  • q.s. to 50 mL with water
  1. Filter sterilize, and aliquot 1 mL into sterile microcentrifuge tubes and store at -20°C
  2. Carbenicillin is commonly used at 50 μg/mL in E. coli cultures.
  3. The presence of carbenicillin in media is indicated by two black lines.
  4. Carbenicillin is abbreviated as "CARB". 

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Kanamycin

30 mg/mL in H2O

To make 50 mL, add:

  • Quantity -1.5 g
  • Chemical - Kanamycin sulfate (FW = 582.58 g/mol)
  • q.s. to 50 mL with water
  1. Filter sterilize, and aliquot 1 mL into sterile microcentrifuge tubes and store at -20°C.
  2. Kanamycin is commonly used at 30 μg/mL in E. coli cultures for plasmid selection. For strains carrying kanamycin resistance on the chromosome, such as Origami B(DE3) or Rosetta-gami B(DE3), use this antibiotic at 15 μg/mL in the media.
  3. The presence of kanamycin in media is indicated by a single red line.
  4. Kanamycin is abbreviated as "KAN".

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Chloramphenicol

34 mg/mL in ethanol

To make 50 mL, add:

  • Quantity - 1.7 g
  • Chemical - Chloramphenicol (FW = 323.13 g/mol)
  • q.s. to 50 mL with 100% ethanol
  1. Store in a 50 mL conical tube at -20°C.
  2. Chloramphenicol is commonly used at 34 μg/mL in E. coli cultures.
  3. The presence of chloramphenicol in media is indicated by a single green line.
  4. Chloramphenicol is abbreviated as "CHLOR".

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Tetracycline

5 mg/mL in ethanol

To make 50 mL, add:

  • Quantity - 250 mg
  • Chemical - Tetracycline (FW = 444.43 g/mol)
  • q.s. to 50 mL with ethanol
  1. Store in a 50 mL conical tube at -20°C.
  2. Tetracycline is commonly used at 12.5 μg/mL in E. coli cultures.
  3. The presence of tetracycline in media is indicated by a single blue line.
  4. Tetracycline is abbreviated as "TET".

Alternatively, if you have tetracycline hydrochloride, make a 10 mg/mL stock in H2O

To make 50 mL, add:

  • Quantity - 0.5 g
  • Chemical - Tetracycline-HCl (FW = 480.90 g/mol)
  • q.s. to 50 mL with water
  1. Filter sterilize, and aliquot 1 mL into sterile microcentrifuge tubes and store at -20°C.
  2. Tetracycline-HCl is commonly used at 11.5 μg/mL in E. coli cultures.
  3. The abbreviation and color indicator are the same for tetracycline and tetracycline-HC.

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Making Competent E.coli Cells

Equipment:

  • 42C water bath
  • 37C incubator (for plates)
  • 37C shaking incubator (for tubes)
  • Autoclaved test tubes with lids
  • Plate spreader
  • Bunsen burner or alcohol lamp
     

Consumable Materials:

  • Culture of DH5a or NEB5a or XL-1 Blues or other E. coli
  • 0.1 M chilled CaCl2
  • LB medium
  • Clean 1.5 mL eppendorf tubes (“epis”)  (or 2 mL epis, doesn’t really matter)
  • Agar plates with selective medium
  • Gas or alcohol for flame sterilizing instruments and aseptic technique
  • Ice bucket or Styrofoam box of wet ice
  1. 1 day before competent cells are needed, start overnight culture of E. coli cells. To do this, inoculate ~2 mLs of LB in a test tube with the E. coli strain and incubate in shaking 37C incubator overnight. To inoculate, take a sterile wooden stick or a sterile p1000 tip, touch to a colony of bacteria from an agar plate, and swirl in the LB broth in the test tube.
  2. The next day, back dilute culture 1:100 or so, such that your cells enter log phase. For example, add 30 uL of the overnight culture to 3 mL fresh LB broth in a new test tube. Place back into incubator.  It will take about 2 or 3 hours for the culture to reach mid-log phase (~0.3 to 0.4 OD600).
  3. Place 0.1 M CaCl2 in fridge to chill while wait, if it is not already stored in the fridge.
  4. Turn on water bath to start warming up to 42C so it is ready when needed (used in step 9).
  5. When the cells have reached mid-log, spin cells down in centrifuge (recommend ~4000 rpm but flexible) to pellet cells. Ideally, perform this step at 4C but the spin is short so not a big deal if spin at room temp.
  6. Remove supernatant and resuspend in 1mL 0.1M CaCl2. Repeat spin.
  7. Perform previous step up to three times, the last time resuspending the pellet in only ~50uL.
  8. Add DNA to cells and incubate on ice for ~30 minutes.
  9. Heat shock by placing tubes in 42C water bath for ~45 sec.
  10. Immediately after, return tubes to ice for 2 min.
  11. Add 500 uL LB/SOC to cells and move to shaking 37C incubator for 1 hour.
  12. Plate dilutions onto agar containing proper antibiotics. Suggest plating 200 uL onto one plate and 20 uL onto another plate for each sample. Can use glass beads or a glass hockey stick to spread the liquid across the surface of the agar plate.
  13. Flip plates upside down (to avoid condensation dripping onto surface of plate) and incubate overnight at 37C.

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MiniPreps

Method 1: DIY style TENS

  1. Transfer 1.5 mL of an overnight culture containing your plasmid to an eppendorf tube and spin at 5000 rpm for 5 min in a tabletop centrifuge to pellet the cells. Discard the supernatant.
  2. Resuspend pellet in either in 50 μL of LB or P1 buffer or TE/RNAse.
  3. Add 300 μL TENS buffer Mix by inverting 5 times.
  4. Add 100 μL 3M NaAc pH 5.2. Mix again.
  5. Spin down at top speed for 5 minutes, Transfer supernatant to a new tube (pouring works).
  6. Add 1 mL 95% to 100% EtOH (ideally ice cold). Spin down at top speed for 5 minutes (ideally at 4˚C).
  7. Wash with 0.5 mL of 70% EtOH. Pour out the supernatant. Spin again to find the rest of the supernatant. Air dry for 10 minutes.
  8. Resuspend the dried pellet in 30 μL of water or TE buffer.

Method 2: DIY style STET

  1. Transfer 1.5 mL of an overnight culture containing your plasmid to an eppendorf tube and spin at 5000 rpm for 5 min in a tabletop centrifuge to pellet the cells. Discard the supernatant.
  2. Add 300 μL STET buffer, and resuspend cells by vortexing.
  3. Add 10 μL lysozyme (10 mg/mL), vortex, and submerge in boiling water for 40 sec.
  4. Spin for 30 min in a tabletop centrifuge at maximum speed at 4 ˚C.
  5. Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.
  6. Add 300 μL ice cold isopropanol to precipitate the DNA.
  7. Spin for 10 min in a tabletop centrifuge at maximum speed at 4 ˚C. Remove supernatant.
  8. Add 200 μL ice cold 80% ethanol to wash pellet and spin for 5 min in a tabletop centrifuge at maximum speed at 4 ˚C. Remove supernatant.
  9. Dry pellet (air dry at room temperature or 37 ˚C or dry in a speedvac).
  10. Rehydrate in 50 μL TE

STET Buffer Recipe (measurements are to make 200mLs)

  • 8% sucrose  (16g)
  • 50 mM Tris-HCl, pH 8  (121.14 g/L * .2 * .05 = 1.21 g)
  • 0.5% Triton X-100   (1 mL)
  • 50 mM EDTA  (372.24g/L * .2 * .05 = 3.722 g)

TE Buffer Recipe

  • 10 mM Tris-HCl, pH 8
  • 1 mM EDTA

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DNA Quantification

Background

Optical density at 260nm (in the UV part of the spectrum) is used to determine concentration of DNA. At 260nm, an OD of 1 is equal to 50 ug/mL of ds DNA. Pure DNA has a ratio of 1.8 A260/A280. Pure RNA has a ratio of 2.0 A260/A280. Low ratios are indicative of protein or phenol contamination.

To measure your sample, you can dilute and read sample in a UV-Vis Spectrophotometer or use 1 to 2 uL of straight sample on a Nanodrop.

  • Standard Qiagen Miniprep amounts: 75 to 300 ng/uL
  • Standard Qiagen Maxiprep amounts (resuspend pellet in .5 mL): 800 to 2000 ng/uL

(Output will vary based on amount of culture used, strain of E. coli used, and the plasmid copy number)

At SoundBio, we are lucky to have a Nanodrop 2000c.  Follow the instructions below to operate:

 

Nanodrop 2000c Instructions

  1. Open laptop and boot up if necessary. This may take a few minutes.
  2. Open the SoundBio Guest account. There is no password for this account.
  3. Launch the software for the nanodrop.
  4. Look in the bottom left corner of the software. If you see a red icon, unplug the instrument USB cord on the front left of the laptop and plug back in after a few seconds. The icon in the bottom left of the software window should be green if the instrument is correctly recognized by the laptop.
  5. Select nucleic acid in the software menu.

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Restriction Enzyme Digest

Digesting a gel-purified PCR product for cloning:

Table1.png

Checking minipreps of clones for the right insert:

 

 

 

 

Table3.png

Ligation Step:

 

 

 

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Glycerol Stocks

LigationTable.png

Sometime we want to be able to store bacteria long-term (weeks to decades). Luckily, we can essentially freeze them down at very low temperatures and have “stock” tubes of the bacteria to store indefinitely. We add glycerol to reduce ice crystals from forming and puncturing the cell membranes. Glycerol is also added to things in everyday life like ice cream to reduce ice crystal formation so the ice cream feels smoother and creamier.

Creating a bacterial glycerol stock:

  1. Locate the freezing media or prepare some. Freezing media is simply regular media (LB for most things) supplemented with 100% glycerol to a final concentration of 15%. You should not use already prepared LB and add glycerol to it since adding the glycerol will dilute the LB.
  2. Start a 2mL overnight culture of the bacteria you want to make a glycerol stock of. Include antibiotics in overnight culture media if necessary (for example, if the strain contains a plasmid).
  3. When the overnight culture has reached a high density (usually ~14 to 24 hours for E. coli), transfer 1.5mL to an eppendorf tube and spin in the tabletop centrifuge for one minute at 9,000 rpm (the speed and time are very flexible, just pellet the cells so they don’t spill out when remove the supernatant).
  4. Open the eppendorf tube and dump the supernatant without losing the cell pellet (just tip upside down slowly / with care).
  5. Locate the cryotubes, take one for each overnight you prepped, and label them well. Standard info to include in label would be the strain, any plasmids it contains, initials of preparer, and date. We often include a number on the top of the tube to reference more detailed info about the sample.
  6. From this point on, try to be efficient about getting it into the freezer. The presence of the glycerol will preserve the cells at -80C, but at room temperature, it will slowly kill off the cells. Therefore, as soon as you add the glycerol-containing media to the cells, don't delay.
  7. Add 1 mL of freezing media (LB+15% glycerol) to the pellet and resuspend. Pipette up and down and/or vortex until well suspended. Failure to resuspend the pellet well at this step is an easy mistake that will have a big effect on your glycerol stock.
  8. Once fully resuspended, transfer to a sterile cryotube (screw cap tube that can withstand the -80C temperatures well).
  9. Place tube in the -80C freezer upright (so that liquid does not freeze against the cap of the tube). Store at -80C indefinitely.

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Starting a Bacterial Culture from a Bacterial Glycerol Stock

DO NOT OPEN THE -80C WITHOUT EXPLICIT PERMISSION

  1. Locate some sterile wooden sticks (autoclaved) and LB agar plates with whatever antibiotics you might need (if the strain contains a plasmid, otherwise plain LB agar plates are fine).
  2. Label the agar plates with strain name, date, your initials, and any other necessary information.
  3. Working quickly, open the -80C door, find the box containing your glycerol stock, and remove that tube. Put the box back in and close the door to the -80C freezer. The -80C freezer should never be open more than 45 seconds (it can be open longer, but the longer, the warmer the inside will get, the more stress the freezer has to return to temp, and the shorter lifespan of everything inside as well as a shorter lifespan for the freezer itself). The shorter the door is open the better.  
  4. Open the cryotube containing your glycerol stock. Try not to set the cap down.
  5. Use the sterile wooden rod to scratch the frozen liquid/ice surface of the tube’s contents. You don’t need to get a large amount of material, but it might take applying some pressure to really scratch into the surface and pick up some bacteria from it.
  6. Gently streak the wooden rod across the surface of the LB agar plate a few times. Do not go back into the glycerol stock. One scratch from the glycerol stock should be MORE than enough for one agar plate.
  7. Cap the glycerol stock. Working quickly, open the -80C freezer, place into same spot you took it from, and close -80C freezer.
  8. If desired, you can perform T-streak using the inoculating loop.

Notes

  • The freezer should never be open long!
  • The stock tubes you pull out should never melt!
  • Work quickly and efficiently, plan ahead so things are in reach and not running around once have the door open or the tube in hand.
  • If you have many glycerol stocks that you are starting strains from, do not pull out more than two or three at once (and if you are new at this, you should only ever work with one at a time). You can streak multiple stocks to one plate, for example, by drawing lines on the bottom of the plate with a sharpie
  • You can scratch some inoculum off the surface and place into liquid media for an overnight instead of a plate, but this should be discouraged. Streaking out onto a plate allows you to select from one colony and ensure genetic fidelity.

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LB+15% Glycerol Recipe

  • 30mL 100% glycerol
  • 2.5 g NaCl
  • 2.5 g Peptone/tryptone
  • 1.25 g Yeast extract

Start by adding roughly half the needed water to a beaker. Add the ingredients and stir until dissolved (it should be easy, no heat needed. If having trouble, just leave it sit for a few minutes and it will dissolve on its own). Add more water to final volume of 250mL. Pour into a 500mL glass bottle and autoclave.

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