Team:CSU Fort Collins/Protocols

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Protocols

PCR

PCR

Materials

  • Template DNA
  • 5X HF Phusion Buffer or 10X HF Taq Buffer
  • Molecular Grade Water
  • dNTPs
  • Primers
  • DNA Polymerase (Phusion or Taq)

Procedure

Piece Amplification

  1. Create the following mixture:
    2. Component 50μl reaction
    Molecular grade H2O Added first. 32.5μL
    5x HF Buffer 10μL
    10 mM dNTPs 1.0μL
    Primer A (10μM) 2.5μL
    Primer B (10μM) 2.5μL
    Template DNA 1.0μL
    DNA polymerase Added last. 0.5μL
  2. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
    3. Cycle Step Temp(C) Time Cycles
    Initial denaturation 98 5 min 1
    Denaturation 98 10s 35
    Annealing LowerTm+3 15s 35
    Extension 72 45s/kb 35
    Final Extension 72 10min 1
    Hold 4 Hold 1

    Colony PCR

    1. Create the following mixture:
    Component 50μl reaction
    Molecular grade H2O Added first 33.5ul
    5x Phusion HF Buffer 10μL
    10 mM dNTPs 1.0μL
    Primer A (10μM) 2.5μL
    Primer B (10μM) 2.5μL
    Colony 1
    Phusion DNA polymerase Added last. 0.5μL
  3. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
    4. Cycle Step Temp(C) Time Cycles
    Initial denaturation 98 5 min 1
    Denaturation 98 10s 35
    Annealing LowerTm+3 15s 35
    Extension 72 45s/kb 35
    Final Extension 72 10min 1
    Hold 4 Hold 1

PCR with Q5

Materials

  • Template DNA
  • Q5 master mix
  • Molecular Grade Water
  • Primers
Component 50μl reaction
Molecular grade H2O Added first. 32.5μL
Q5 Master Mix 25μL
Primer A (10μM) 2.5μL
Primer B (10μM) 2.5μL
Template DNA 1.0μL
  • Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
    • Cycle Step Temp(C) Time Cycles
    Initial denaturation 98 5 min 1
    Denaturation 98 10s 35
    Annealing LowerTm+3 15s 35
    Extension 72 30s/kb 35
    Final Extension 72 10min 1
    Hold 4 Hold 1

    Colony PCR

    1. Create the following mixture:
      2. Component 50μl reaction
      Molecular grade H2O Added first 32.5ul
      Q5 Master Mix 25μL
      Primer A (10μM) 2.5μL
      Primer B (10μM) 2.5μL
      Colony 1
    2. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
      3. Cycle Step Temp(C) Time Cycles
      Initial denaturation 98 5 min 1
      Denaturation 98 10s 35
      Annealing LowerTm+3 15s 35
      Extension 72 30s/kb 35
      Final Extension 72 10min 1
      Hold 4 Hold 1


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    Digestion

    Digestion

    Materials

    • 2 LB + Antibiotic plates
    • Spreaders or glass beads
    • LB Media
    • Ligated back bone
    • Digested back bone(control)

    Procedure

    1. Create the following mixture:
    Component 50μl reaction
    Molecular Grade Water Calculated. Use to make reaction total volume = 50ul
    Custsmart Buffer(different if using pstI) 5ul
    DNA 1ug
    Enzyme 1 .5ul
    Enzyme 2 0.5ul

    2. Digest your mixture in a water bath at 37C for 60min



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    Ligation

    Ligation

    Ligation Using the “Ligation Template” excel sheet, calculate the amount of each component to combine in a ligation mix for an Insert: Backbone ratio of 4:1

    Component 20μl reaction
    Molecular Grade Water Calculated. Use to make reaction total volume = 20ul
    T4 DNA Ligase Buffer 2ul
    Insert DNA
    Backbone DNA
    T4 DNA Ligase 1ul

    Incubate at 16C overnight on a heating block or at room temperature for 1 hour.



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    Transformation

    Transformation

    Materials

    • 2 LB + Antibiotic plates
    • Spreaders or glass beads
    • LB Media
    • Ligated back bone
    • Digested back bone(control)

    Procedure

    1. Set water bath to 42C
    2. Remove LB+Antibiotic plates from 4C and allow them to come to RT.
    3. Thaw chemically competent cells on ice. Leave in microcentrifuge tube.
    4. Add 5μL of ligation mix chemically competent cells.
    5. Add 5uL of digested back bone to chemical competent cells as a control
    6. Incubate on ice for 30 min.
    7. Heat shock cells for 60 sec at 42C without shaking.
    8. Aseptically (by the fire or in the hood) add 250μL of LB media to the tube (DO NOT ADD ANTIBIOTIC AT THIS STEP). Cap tightly.
    9. Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.
    10. In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.
    11. Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.


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    LB Media and Plates

    LB Media

    Materials

    • LB Miller (Powder)
    • 1L Glass Bottle
    • Stir Bar
    • DI/RO Water

    Procedure

    1. Triple rinse bottle with DI water
    2. Add stir bar to the bottle
    3. Add 700 mL of DI Water to the 1L Bottle
    4. Add 17.5 g of LB Miller
    5. Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
    6. Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
    7. Place autoclave tape on top
    8. Autoclave on Liq 30
    9. Let cool and then tighten the cap and store at room temperature

    LB Agar Media for Plates

    Materials

    • LB Miller (Powder)
    • 1L Glass Bottle
    • Stir Bar
    • DI/RO Water
    • Agar
    • Antibiotic if applicable
    • Petri Dish Plates
    • Ethanol Proof Markers
    • Tape

    Procedure

    1. Triple rinse bottle with DI water
    2. Add stir bar to the bottle
    3. Add 700 mL of DI Water to the 1L Bottle
    4. Add 17.5 g of LB Miller
    5. Add 8.4g of agar (not agarose)
    6. Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
    7. Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
    8. Place autoclave tape on top
    9. Autoclave on Liq 30
    10. Remove IMMEDIATELY from autoclave when finished
    11. Place on stir plate and slowly stir (no air bubbles from stirring too fast) until the bottle can be held comfortable for a 7 seconds (20-30 min of cool down time)
    12. Add 700 μL of aliquoted Antibiotic
    13. INSIDE THE HOOD:
      1. Spray the sleeve of plates, the agar media, the marker, and the tape down with ethanol before putting them in the hood. When opening the sleeve of plates, be careful to not rip it because you will put the plates back into it.
      2. Following sterile technique pour the plates about a 3rd of the way full
      3. Allow to cool in the hood until they have solidified and are no longer warm
      4. Turn the plates upside down and put back in the sleeve
      5. Tape the sleeve shut and write the antibiotic and the date they were made on the tape.


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    Gel Electrophoresis

    Gel Electrophoresis

    Materials

    • Agarose
    • 1X TAE Buffer
    • Parafilm
    • Electrophoresis chamber and power source
    • SYBR Green
    • Loading Dye
    • DNA Ladder
    • PCR Samples

    Procedure

    1. Using a balance, mass out 0.5 gram of agarose (for 1% gel, for 2% mass out 1.0 gram). Mix this with 50 mL of TAE 1X Buffer.
    2. Microwave the mixture, mixing by swirling intermittently, until all the agarose is dissolved and the mixture is homogeneous and clear (about 2 minutes).
    3. Pour the mixture into a gel plate and insert a correctly-sized comb (8, 10, etc. lanes). Allow to cool and solidify on the benchtop.
    4. Mix your samples on a piece of parafilm. Use 10 μL of the appropriate DNA Ladder (1 kb, 100 bp, 2 log, etc.) combined with 1 μL SYBR Green/DMSO in the first lane. For all samples, mix 10 μl of sample and 2 μL of SYBR Green/Dye Mixture.
    5. Place gel in tray into the gel electrophoresis apparatus - wells should be close to the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill - there is a guide sticker on the apparatus which marks the max fill line.
    6. Load 10 μL of each sample into the appropriate wells.
    7. Connect wiring and run gel electrophoresis at 110 V for 1 hour.
    8. Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.


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    Phosphorylation & Ligase Cycling

    Phosphorylation

    Materials

    • PCR product that are to being ligated
    • Ampligase thermostable DNA ligase reaction buffer
    • T4 polynucleotide kinase[10 U/μL]
    • 10 mM ATP
    • Molecular Grade Water

    Procedure

    1. Make the following reaction mixture.
      2. Component 20μl reaction
      Molecular Grade Water Calculated. Use to make reaction total volume = 20ul
      PCR products 90 fmol each
      Ampligase reaction buffer 2 μL
      T4 polynucleotide kinase 1 μL
      10 mM ATP 2 μL
    2. Incubate reaction at 37 C for 1 hour.
    3. Heat inactivate reaction at 65 C for 20 minutes.

    Ligase cycling reaction

    Materials

    • Phosphorylated mixture
    • Bridging oligonucleotide
    • Ampligase thermostable DNA ligase reaction buffer
    • Ampligase thermostable DNA ligase
    • Molecular Grade Water

    Procedure

    1. Make a mixture of all bridging oligonucleotide, by adding 10 μL of each 100 μM bridging oligonucleotide stock and then adding Molecular Grade Water until 1000 μL.
    2. Make the following reaction mixture.
      3. Component 10μl reaction
      Molecular Grade Water Calculated. Use to make reaction total volume = 15ul
      Phosphorylated mixture 11.60μl
      Ampligase reaction buffer 1.5 μL
      Bridge mixture 1 μL
      Ampligase .9 μL
    3. Use thermocycler at folowing settings.
      4. Cycle Step Temp(C) Time Cycles
      Initial denaturation 94 2 min 1
      Denaturation 94 10s 25
      Annealing 55 30s 25
      Extension 66 60s/kb 25
      Hold 4 Hold 1
    4. PCR products are ready to be transformed.


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    Chemically Competent E. coli Cells

    Chemically Competent E. coli Cells

    Media to Prepare

    • At least 100 mL of LB
    • At least 40 mL of 100 mM CaCl2
    • At least 4 mL of 100 mM CaCl2 + 15% glycerol
    • *Autoclave or filter sterilize each solution
    • **Autoclave 2- 250 mL flasks with foam stoppers and some microcentrifuge tubes

    Procedure

      End of Day 1

    1. Start a overnight (O.N) culture of E. coli cells in 5 mL of LB (usually from a single colony on a plate) .

      2. Beginning of Day 2

    2. Inoculate 100 μL of the O.N. culture of E. coli into 50 mL LB in 250 mL shake flask
    3. Grow at 37 C until OD reaches 0.4 to 0.6
    4. Place tubes on ice for 30 min, mixing periodically to ensure uniform cooling
    5. Centrifuge for 5 min at 4 C and 3000 rpm [important for rotor to be cold before starting]
    6. Decant supernatant
    7. Centrifuge for 5 min at 4 C resuspend gently in 10 mL of ice cold 100 mM CaCl2
    8. Place on ice for at least 30 min [2 hours is optimal and 3000 rpm [important for rotor to be cold before starting]
    9. Decant supernatant and resuspend gently in 1 mL of ice cold 100 mM CaCl2 + 15% glycerol
    10. Place on ice overnight [put ice bucket at 4 C so the ice doesn’t completely melt]

      11. Day 3

    11. Ready for transformation or for 100 μL aliquots into cold microcentrifuge tubes which can be flash frozen and stored at -80 C



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    BG-11 Media Preparation Protocol

    BG-11 MEDIA PREPARATION PROTOCOL

    Part I: Preparing BG-11 Components A, B, C, and Trace

    Part II: Preparing BG-11 liquid media

    Part III: Preparing BG-11 agar plates

    Part I: Preparing BG-11 Components A, B, C, and Trace

    1. Label three 500mL bottles as BG-11 A (100X), BG-11 B (600X), and BG-11 C (100X)
    2. Label one 250mL bottle as BG-11 Trace (1000X)
    3. Make media components
      4. Part Chemical name Amount
      Part A(100X) Sodium nitrate (in nitrate box on top shelf) 30.000g
      Part A(100X) Magnesium sulfate heptahydrate 1.498g
      Part A(100X) CaCl2-2H2O Calcium chloride dehydrate 0.720g
      Part A(100X) Citric acid 0.120g
      Part A(100X) Ammonium ferric citrate 0.120g
      Part A(100X) IDRANAL 0.020g
      Part B(600X) Potassium phosphate trihydrate 4.8g for 600X (0.800g for 100X)
      Part C(600X) Sodium carbonate 0.400g
      Trace Metals(1000X) Boric acid 0.572g
      Trace Metals(1000X) Manganese II chloride Tetrahydrate 0.362g
      Trace Metals(1000X) Zinc sulfate heptahydrate 0.044g
      Trace Metals(1000X) Sodium molybdate dehydrate or molybolic acid disodium salt dehydrate 0.078g
      Trace Metals(1000X) Copper II sulfate pentahydrate 0.016g
      Trace Metals(1000X) Cobalt II nitrate hexahydrate 0.010g
    4. Add 200mL of DI H2O to each bottle
    5. Autoclave ONLY Parts A, B, and C (NOT TRACE) on Liquid Cycle, 30-45min
    6. Filter sterilize Trace metals
    7. Store all components at 4°C

    Part I: Preparing BG-11 Components A, B, C, and Trace

    1. Autoclave 700mL of DI H2O in a 1L bottle
    2. In sterilized laminar flow hood
      1. Add 7mL of BG-11 Component A
      2. Add 7mL of BG-11 Component B
      3. Add 7mL of BG-11 Component C
      4. Add 700µL of BG-11 Component Trace Metals
      5. pH adjust to 8.0±0.05 with NaOH and HCl
      6. Once adjusted to pH 8.0, add 7mL of 1M TES-NaOH
      7. Add antibiotics as needed
    3. Store media at 4°C or room temperature



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    BG-11 Agar Plates

    BG-11 Agar Plates

    Materials

    • BG-11 component A 100X solution
    • BG-11 component B 100X solution
    • BG-11 component C 100X solution
    • Agar
    • 1 L glass bottle
      • Stir bar
    • 3 M NaOH
    • 1 M Tes-NaOH (pH 8.0)
    • Trace metals mix 1000X solution
    • Antibiotics (optional)
    • 1 sleeve of plates (25)

    Procedure

    1. Dissolve 7 g of Agar powder in 700 mL of deionized water in a 1 L glass bottle with blue cap (will make enough for 1 sleeve of plates).
    2. Place a piece of autoclave tape over the orange cap and onto the neck of the glass bottle. Label the bottle (media, initials, date)
    3. Autoclave the mixture in the bottle on the liquids cycle (3 or 4). See Sterile Technique and Autoclaving Protocol for more details. This procedure takes 45-60 minutes
    4. While cooling, put the bottle on the stir plate and stir the agar to prevent it from hardening prematurely. Allow the liquid agar to cool so that you can comfortably grab the bottle with a bare hand.
    5. Once you are able to comfortably grab the bottle for >5 sec with your bare hand, add 7ml each of BG-11 components A, B, and C 100X. Then add 700μL of trace metals mix 1000X.
    6. pH adjust the mixture to pH of 9.0 with 3 M NaOH (use pH meter in GL 122; add approximately 10μL at a time ). Once adjusted, lock in the pH by adding 7ml of 1M Tes-NaOH. Swirl the bottle periodically to ensure proper mixing of all components.
    7. [Optional] Add antibiotics as needed (Kanamycin, refer to table below). Allow to stir on stir plate for 1 min to mix the antibiotic with the media and to reduce bubble formation.
      8. Type of BG-11 Kan plate Volume of Kanamycin stock (50 mg/ml) to add to 700ml BG-11 mixture
      BG-11 + Kan 10 (10µg/ml Kan) 140µL
      BG-11 + Kan 20 (20µg/ml Kan) 280µL
      BG-11 + Kan 30 (30µg/ml Kan) 420µL
      BG-11 + Kan 40 (40µg/ml Kan) 560µL
      BG-11 + Kan 50 (50µg/ml Kan) 700µL
    8. Meanwhile, spray down a sleeve of plates with 70% ethanol and place in laminar flow hood. Open the sleeve and remove plates.
    9. Starting at the bottom of the stack, open each plate and pour in agar until the bottom of the plate is covered. Typically, 18-20 mL of agar is used per plate. Replace the stack on top of the poured plate.
    10. Remove the next cover. Repeat step until you have poured all the plates. Label the plates with the appropriate colored lines on the sides of each plate.
    11. Once the plates have set (5-15 min), turn the plates over. The plates should sit like this for around 1 hour until they have completely cooled.
    12. Place the plates back into the plastic with the agar side on top (upside down), seal and label bag (media, initials, date), store at 4C


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    Concentrated transformation techniques for Synechocystis sp. PCC 6803

    Concentrated transformation techniques for Synechocystis sp. PCC 6803

    1. Natural uptake, and transformation on BG-11
      1. Grow cells to an OD (730 nm) of 0.5-0.75
      2. Centrifuge 10 ml cells at 4000 g for 6 min. Discard supernatant.
      3. Re-suspend cells in 100 microliters of BG-11 and move suspension to
        1. 14 ml conical falcon tube.
        2. Add 1 microgram of target plasmid to the suspension
        3. Also prepare 1 control tube with no DNA
      4. Incubate cells for 5 hrs in light at 30+ C.
        1. Tubes should be gently shaken at ~2.5 hrs to ensure proper mixing
      5. Plate/spread 100 uL of the mixture onto the following plate
        1. BG-11 (no-antibiotics)- Add 100ul cell and plasmid suspension directly to the agar. To ensure complete spreading and full contact between the cells and agar, use a bacterial spreader.
        2. After 48 hours use the bacterial loop spreader to collect a smear of cells from the BG-11 (no-antibiotics) plate. Perform a triple streak procedure onto a BG-11+Kan50 plate
        3. Cover plates with surgical tape and PLACE all plates in 30+ C lighted incubator.
      6. Single colonies should be observed in 10-14 days.
      7. After colonies have formed, Streak single colonies on a fresh BG-11 plate with antibiotics and place back into incubator to grow. Most protocols do this once a week for 3-4 weeks to ensure segregation of the mutation.


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    Transformation of mazF KmR (NF) strains (2nd transformation)

    Transformation of mazF KmR (NF) strains (2nd transformation)

    Transformation

    1. Grow engineered cells to an O.D of 0.5 to 0.7.
    2. Centrifuge 2x 10mL of cells at 4000x g for 6 minutes at room temperature. Discard supernatant.
    3. Resuspend cells in 100µL of BG-11 and move suspension to 14ml falcon tube.
    4. Add 1µg of plasmid to each culture to be transformed. Make sure one culture has no plasmid (as a control). Note: Try to achieve a mixture with 10ng/μL of DNA concentration.
    5. Incubate cells for 5 hours in light at 30oC. Shake gently at 2.5 hrs to ensure proper mixing.
    6. After 5 hours, spin down cells and resuspend in 10mL of BG-11. Transfer the culture to a shake flask. Leave to grow for 24 hours.
    7. Plate 100µL of each culture on BG-11+Ni 5.3 (20μM Ni) plates.
    8. Cover plates with surgical tape and leave it to grow under light at 30C
    9. Colonies should appear in 5 to 7 days.
    10. Once colonies form, streak single colonies on both BG-11+Ni5.3 and BG11+Kan50 plates. Note he colonies with the expected insert should survive on BG-11+Ni5.3 but NOT on BG-11+Kan50.
    11. Transfer to liquid cultures
    12. In a 14mL falcon tube, take a swab of cells from the BG-11+Ni5.3 plate and place it in 1mL of BG-11+20μM Ni. Let it grow for 2-3 days. Note: To achieve 20µM Ni, add 0.2µL of 100mM NiSO4 per 1mL BG-11.
    13. Once cells reach a sufficient O.D in the tubes (about 2 days), pipette all of the cells into a 50mL BG-11+20μM Ni culture. Cells should be retransferred every 6 to 7 days. Note: To achieve 20μM Ni, add 10μL of 100mM Ni to 50mL of BG-11.


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