Team:USAFA/Experiments

 

Experiments

Basic Lab Prep and Growth Procedures

Making LB

  1. Read the directions on the bottle . For example, to make LB Agar. The bottle says to add 40 grams off the LB powder per liter of distilled water. (Note: the gram amount may differ for each bottle. See bottle for instructions).
  2. Scale 12.5 grams of LB powder of 500 mL of distilled water with graduated cylinder.
  3. Always heat the mixture to dissolve fully using a heat plate and a flask. Use an autoclaved flask. Make sure to have stir bir to dissolve particles. Black autoclaved tape on the flask means that it has been autoclaved and is sterile. Temperature doesn't matter. Dark urine color.
  4. Autoclaved for 15 minutes in 121 degrees. TIghten media bottle cap . Put foil on top of lid and put autoclave tape. Use double volume bottle for however much liquid mixture being autoclaved. For example, use a liter bottle for a 500 mL mixture.
  5. Tip when labelling with masking tape, flip end of one tape so easy to pull off.
  6. Always triple rinse glassware with DI water after washing with soap and warm water.

Bacterial Plating

  1. Add 5 mL of LB broth to each test tube (cultured tubes) (3 of them).
  2. From the bacteria plate, take a distinct colony for each test tube and swirl it into the LB broth.
  3. Plate two more plates with the original plate. Streak on one side of the plate, do a quarter turn, and then overlap the streaks at this direction.
  4. Placed the plates in the incubator and ensure that all surfaces are cleaned. The temperature will vary based on different bacteria.

Making Gel for Electrophoresis

(For 50ml of 2% agarose gel)

  1. Add 1 gram of iGEM agarose (B/c 2% of 50 is 1)
  2. Add 50 ml of 1x tbe ( a bit more over 50 is okay due to evapoation) and mix
  3. Stuff flask with tissue
  4. Microwave for 30 seconds- stop when mix starts to boil to avoid over flowing, start again when solution settles
  5. OPTIONAL, either mix in GelGreen or GelRed, or dye with EtBr later after Electrophoresis ran.
  6. Continue to microwave until there are no more agarose crystals left. Make sure the ENTIRE solution is clear, Look meticulously.
  7. Once it is clear then it can be poured.
  8. Let it cool in the fridge for about 10mins, or until opaque.

Gel Preparation(Summer procedure)

Make a gel buffer, then add agarose.

  1. Obtain a 1 liter container. Add 20 mL of buffer and fill the rest of the container with 18 miliohm water until 1 liter volume is attained.
  2. Pour 50 mL of the solution into a smaller flask. Add 0.4 g of agarose powder. Microwave mixture for about a minute and a half (you can allow it to boil for 20-30 sec).
  3. When the flask can be held comfortably for 5-6 sec, add 5 microliters of ethidium bromide and swirl to mix. Pour mixture into gel container and insert the comb (this makes the wells) onto the side where the DNA and ladders will be inserted. The gel will solidify in about 20 min.
  4. Pull comb out of solidified gel. Plug in power source and hook wires to gel electrophoresis instrument ensuring that the DNA will flow from negative to positive.
  5. Pour buffer into gel electrophoresis instrument so that a thin layer covers the top of the gel.
  6. Add 5 microliters of each sample, keeping track of what well each sample is insterted into.
  7. Set power to 130-140 V for about a half hour or until the DNA has moved about halfway or 3/4 of the way down the plate.

How to Grow & Preserve Rhodococcus RHA1 in Glycerol for Freezing

  1. Culture bacteria in LB broth in incubating orbital minishaker at 30 degrees C and 250rpm for 48 h.
  2. Under biosafety cabinet (hood), add 500 mL of Rhodococcus RHA1 in broth to Corning cryotube
  3. Under hood, add 500mL 50% sterile glycerol mix (See below if you need to make this)
  4. Place tubes into cryo Freezing Container
  5. Place in -80 Freezer
  6. Next day, take out of -80 freezer and put into a box in the -80 freezer.

How to Prepare Pseudomonnus aeruginosa in Glycerol for Freezing

  1. Culture bacteria in LB broth in incubating orbital mini shaker. Set to 37 degrees C and 250 rpm for 48hrs.
  2. 5mL LB broth into cryotube
  3. Obtain single colony with wire loop. Swirl it around in tube.
  4. Place tubes into cryo Freezing Container
  5. Place in -80 Freezer
  6. Next day, take out of -80 freezer and put into a box in the -80 freezer.

Making Standard Solution Cultures for a PFOA Kill Curve for Pseudomonnus aeruginosa and Rhodococcus

  1. Used the cultures made on Monday 8 April
  2. Added ~5 ml LB broth to 10 test tubes (5 for each strain)
  3. Concentrations: 250uM, 125uM, 62.5uM, 31.25uM, control
  4. Add 250uL PFOA into 10mL of LB broth.
  5. From that 10mL test tube, create subsequent standard solutions. Cut in half each time (Take 5mL from the original 10mL PFOA solution, add that to 125uM tube, mix, suck 5mL back into same pipette. Bring that 5mL to the next tube (62.5mM) and do same thing. Repeat for 31.25uM and left one tube as a control. (Then do again for the next 5 tubes)
  6. Micropipette 50uL of bacteria into each tube. (Pseudomonnus aeruginosa in 5, Rhodococcus in 5)
  7. Incubate Pseudomonnus cultures at 37 degrees C in regular incubator for 24 hours and Rhodococcus cultures at 28 degrees C in shaker incubator for 48 hours

WRIGHT PATT: Transformations

Gibbson Assembly: 4-6 fragment method

  1. 10 microliters of DNA Assembly Master Mix, 10 microliters of di-water for a total volume of 20 microliters.
  2. Incubated the Gibson assembled sample at 50 deg C for 60 min.
  3. After incubation, transformation into E. coli was attempted.
  4. The chemically-competent cells were thawed on ice.
  5. Two microliters of the Gibson assembled product was added to the competent cells.
  6. The tube was flicked 4-5 times to mix the components.
  7. The mixture was placed on ice for 10 min and then heat shocked at 42 deg C for 30 sec.
  8. It was then transferred immediately to ice for 2 min.
  9. A volume of 950 microliters of chilled SOC media was added to the mixture.
  10. The mixture was then incubated ar 37 deg C for 60 min (shaken in incubator).
  11. One hundred microliters of cells were spread onto a selection plate after incubation.
  12. The plate was left on the bench to grow in room temperature over the weekend.

Transfromation:

A 1.5 mL vile of e. coli NEB5-alpha cells frozen at -80were thawed on ice (not at room temperature) for ~15 minutes.

  1. Add 3 of the 6 uL of the TOPO Cloning Reaction (from Monday, 6/3) to a vial of thawed, chemically competent e. coli NEB5-alpha cells. Do not mix by pippetting up and down. (Instead, flick it a few times and put it back on the ice)
  2. Incubate on ice for 30-45 minutes.
  3. Heat-shock the cells for 30 seconds at 43 degrees C without shaking. (water bath)
  4. Add 250 uL of room temperature S.O.C. medium.
  5. Cap the tube tightly and shake the tube horizontallhy (200 rpm) at 37 degrees C for 1 hour.
  6. Spread 10-50uL from each transformation on a prewarmed selective plate and incubate overnight. To ensure even spreadging of small volumes, add 20 uL of S.O.C. medium. It is recommended that two different volumes are plated to ensure that at least one will have well0-spaced colonies. (We 10uL on one plate, 50 uL on antother, and the remaining ~190uL on another plate and labelled them accordingly).
  7. After incubation, pick 4-10 colonies for analysis.

DNA+RNA Prep

Digestion

  1. For digestion, use 1-5 micrograms of DNA, buffer at 10x concentration
  2. 1 unit of enzyme per microgram of DNA that can be digested in 1 hour
  3. Fill the rest of the desired volume with 18 miliohm water.
  4. Total volume shouldn't exceed 60 microliters.

Tradition Ligation

  1. 7 ul vector (~10.7 ng/ul)
  2. 10 ul Insert (`10 ng/ul)
  3. 1 ul Enzyme (T4)
  4. 2 ul Buffer (T4)
  5. Mix all four together (in order of descending volume) then let sit at room temp (or 16 degrees celsius) overnight.

qRT-PCR

  1. For each sample, weigh 25–50 mg acid-washed glass beads (150–600 μm diameter) in a 2 ml Safe-Lock tube (not supplied) for use in step 4.
  2. Harvest bacteria by centrifuging at 5000 x g for 5 min at 4ºC (do not use more than 1 x 109 bacteria). Decant the supernatant, and carefully remove any remaining media by aspiration. If the centrifuge will be used later in this procedure, heat it to 20–25ºC. Note: Incomplete removal of medium will inhibit lysis and dilute the lysate, affecting the conditions for binding of RNA to the RNeasy membrane. Both effects may reduce RNA yield.
  3. Add the appropriate volume of Buffer RLT (see table below). Vortex vigorously for 5– 10 s. 9. Note: Ensure that β-ME is added to Buffer RLT before use (see “Important points before starting”). Ensure that the pellet is thoroughly resuspended in Buffer RLT
  4. Transfer the suspension into the 2 ml Safe-Lock tube containing the acid-washed beads prepared in step 1. Disrupt cells in the TissueLyser for 5 min at maximum speed. Other methods of mechanical disruption can be used instead. We recommend using the TissueLyser
  5. Centrifuge for 10 s at maximum speed. Transfer supernatant into a new tube (not supplied). The volume of the tube must be at least twice that of the Buffer RLT used.
  6. Determine the volume of supernatant. Add an equal volume of ethanol (70%), and mix well by pipetting. Do not centrifuge. After adding ethanol, a precipitate may form. This will not affect the RNeasy procedure.
  7. Transfer up to 700 μl lysate, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.* Reuse the collection tube in step 8. If the lysate exceeds 700 μl, centrifuge successive aliquots through the spin column. Discard the flow-through after each centrifugation.*
  8. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.* Reuse the collection tube in step 9. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Be sure to empty the collection tube completely. Skip this step if performing optional on-column DNase digestion.
  9. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flowthrough. Reuse the collection tube in step 10. Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use (see “Important points before starting”).
  10. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.
  11. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid gently, and centrifuge at full speed for 1 min. Perform this step to eliminate any possible carryover of Buffer RPE, or if residual flow-through remains on the outside of the RNeasy spin column after step 10.
  12. If the expected RNA yield is >30 μg, repeat step 12 using another 30–50 μl RNase-free water, or using the eluate from step 12 (if high RNA concentration is required). Reuse the collection tube from step 12. If using the eluate from step 12, the RNA yield will be 15–30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.

QIAquick PCR Purification

  1. Used for purification of up to 10 ug of PCR product (10 - 100 bp) - (Used for G block)
  2. Add 5 volumes of PB Buffer to 1 volume of PCR product and mix. If color orange or violet add 10 ul of 3 M Sodium Acetate, pH 5.0. Mixture should turn yellow
  3. place a QIAquick column in provided 2 ml collection tube.
  4. To bind DNA apply sample to QUIquick column (centrifuge at 13000 rpm for 30-60 second). Discard flow through and insert column back in the same tube
  5. Wash - add 750 ul PE Buffer to QIAquick column (centrifuge at 13000 rpm for 30-60 second). Discard Flow through. and insert column back in the same tube.
  6. Centrifuge QIAquick column once more in provided 20 ml collection tube to remove residual wash buffer.
  7. Place each column in clean 1.5 ml microcentrifuge tubes. (let dry for a minute due to ethanol in misture).
  8. Elution - add 50 ul EB buffer or water to the center of the QIAquick membrane and centrifuge for 1 minute.
  9. If analyzing Purified DNA on gel add 1 volume loading dye to 5 volumes of purified DNA.

QIAquick Gel Extraction Kit

  1. Used for purifying of up to 10 ug of DNA from gel.
  2. Cut the DNA from the agarose gel
  3. Weigh gel in colorless tube. Add 3 volumes QC Buffer per 1 volume gel (gel ~ 100 mg = 10 ul).
  4. Incubate tube 50 - 60 degrees for 10 minutes (or until gel completely dissolved). Vortex every 2 - 3 minutes to break down gel. If color orange or violet add 10 ul of 3 M Sodium Acetate, pH 5.0. Mixture should turn yellow
  5. Place sample in 2 ml collection tube. Centifuge for 1 minute or until all sample passes through column. Discard flow through.
  6. Wash - add 750 ul PE Buffer to QIAquick column (centrifuge at 13000 rpm for 30-60 second). Discard Flow through. and insert column back in the same tube.
  7. Repeat step 5
  8. Place each column in clean 1.5 ml microcentrifuge tubes. (let dry for a minute due to ethanol in misture).
  9. Elution - add 50 ul EB buffer or water to the center of the QIAquick membrane and centrifuge for 1 minute.
  10. If analyzing Purified DNA on gel add 1 volume loading dye to 5 volumes of purified DNA.

MINIPREP

  1. Pellet 5 mL of culture into each Epindorf tube, 6 tubes total. Add 2 mL at a time, only adding 1 mL in the last addition. Spin down tube in centrifuge after each 2 or 1 mL addition.
  2. Resuspend pellet with 250 microliters of Cell Resuspension Solution. Do not vortex after resuspension.
  3. Add 250 microliters of lysis solution to each sample; invert 4 times to mix.
  4. Add 10 microliters of the protease solution to the sample; invert 4 times to mix. Incubate at room temperature for 5 min.
  5. Add 350 microliters of Neutralization Solution; invert 4 times to mix.
  6. Centrifuge at top speed for 10 minutes at room temperature.
  7. Insert spin column into collection tube.
  8. Decant cleared lysate into Spin Column.
  9. Cetrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert column into collection tube.
  10. Add 750 microliters of Wash Solution. Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into collection tube.
  11. Repeat step 10 with 250 microliters of wash solution.
  12. Centrifuge at top speed for 2 minutes at room temperature. Let sit to dry 4-5 min.
  13. Transfer spin column to a 1.5 mL microcentrifuge tube.
  14. Add 100 microliters of nuclease-free water to the spin column. Centrifuge at top speed for 1 minute at room temperature. Let sit for 1 min to allow for better efficiency of elution.
  15. Discard column and store DNA at -20 deg C. If in multiple tubes, combine liquid from all tubes into one.

MINIPREP (promega)

  1. Pellet 8 mL of culture into 5 Epindorf tube, using an additional counterbalance tube (6 tubes total). Add 2 mL at a time, then spin at 14000 rpm for 1 minute after each. (Add 2 mL, spin, dump, add 2 more.. etc). After last spin, decant lysate and remove any remaining fluid near the pellet with a micropipette.
  2. Thoroughly resuspend pellet with 250 microliters of Cell Resuspension Solution. Do not vortex after resuspension.
  3. Add 250 microliters of lysis solution to each sample; forcibly invert 4 times to mix.
  4. Add 10 microliters of the protease solution to the sample; forcibly invert 4 times to mix. Incubate at room temperature for 5 min.
  5. Add 350 microliters of Neutralization Solution; forcibly invert 4 times to mix.
  6. Centrifuge at top speed (1400 rpm) for 10 minutes at room temperature. Binding of Plasmid DNA
  7. Insert spin column into collection tube.
  8. Decant cleared lysate into Spin Column.
  9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert column into collection tube.
    10. Washing
  10. Add 750 microliters of Wash Solution. Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into collection tube.
  11. Repeat step 10 with 250 microliters of wash solution.
  12. Centrifuge at top speed for 2 minutes at room temperature. Let sit to dry 4-5 min.
  13. Transfer spin column to a 1.5 mL microcentrifuge tube.
  14. Let column sit 3-5 minutes to allow ethanol to evaporate out.
  15. Add 100 microliters of nuclease-free water to the spin column. Centrifuge at top speed for 1 minute at room temperature. Let sit for 1 min to allow for better efficiency of elution. -Discard columns (keep flowthrough) and digest samples with Eco RI.

Cloning

prmA-mRFP cloning into pSB1C-mRFP

  1. Mini-prep pSB1C3-mRFP (from Mach 1 cells). Use lysate from 6/mL column.
  2. Digest pSB1C3-mRFP (digest 1-5 uL of DNA) and prmA-mRFP gBlock with Eco RI and Pstl (double digest). Use CutSmart Buffer.
  3. Run digestion of pSB1C3-mRFP out on 0.8? TAE agarose (with 1kbPlus ladder). Cut out the vector band (~2kb) and extract DNA from the gel.
  4. Use PCR cleanup kit to clean up the gBlock digestion.
  5. Run 5 uL of purified vector and gBlock out on 0.8% agarose gel to verify presence of DNA.
  6. Set up ligation reaction with T4 DNA ligase (overnight ligation at 16 degrees C).
  7. Transform ligation reaction into NEB5-alpha cells & screen colonies.
  8. Culture selected colonies, digest with EcoRI and Pstl, and perform gel electrophoresis.

Topo Cloning of prmA-mRFP gBlock

  1. TOPO colone prmA-mRFP gBlock using TOPO Blunt kit. The plasmid has a kanamycin (Kan) resistant cassette.
  2. Transform 3 L TOPO ligation reaction into NEB5-alpha cells.
  3. Screen NEB5-alpha/pTOPO-Blunt-prmA-mRFP gBlock clones.

Topo Cloning of GBlock

Using Zero Blunt TOPO PCR Cloniing Kit, mix the following together (in order of descending volume) in an 1.5 mL eppendorf tube.

  1. 0.5-4 uL gBlock (2 uL were used this time)
  2. 1uL salt solution
  3. 1uL PCR Blunt II-TOPO
  4. add 18 ohm sterile water to a total of 5uL (1uL was used this time)
  5. Final Volume: 6uL

To ensure minute amounts of liqud are mixing, pippete up and down a few drops. The small centrifuge can also be used for a couple seconds to ensure liquid goes to the bottom of the eppindorm tube.

Incubate at room temperature for 30 minutes. Freeze at -20 degrees Celsius to store.