Team:CSU Fort Collins/Notebook/Protocols=Cloning

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    <h2>CLONING A GENE INTO A PLASMID</h2>
 
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Revision as of 15:30, 9 October 2014

Cloning a Gene into a Plasmid

Cloning a Gene Into a Plasmid

Show Table of Contents

Design Primers

  1. Locate Genetic Sequence of Interest
    • Check: do you need to add a restriction enzyme site to the primers to aide in contruction?
    • Other considerations:
    • Primer length should be between 18-30 base pairs (bp)
    • Avoid repeats of 4 or more
    • The melting temperature (Tm) of the two primers which amplify one gene should be close together and between 52 °C and 65 °C
    • At the 3' end, you should end with a G or C, avoid a T or mismatches, and avoid runs of 3 or more G/Cs in the last 5 bps at the 3' end
    • G/C content should be 40-50%

PCR Amplification of Gene

  1. Isolate DNA to be amplified
    • If DNA is located on a plasmid in E. coli, use a plasmid miniprep kit to isolate plasmid DNA (follow kit instructions) from an overnight culture of backteria
    • If DNA is located in the genome of some organism, do the appropriate genomic DNA extraction
  2. PCR amplification
    • Once you receive primers:
    • Spin vials for 5 minutes at maximum speed in the microcentrifuge
    • Make a 100 μM stock solution using molecular grade (nuclease-free) water
    • Vortex for at least 1 minute
    • Dilute stock solution 1:10 fold for a working solution of 10 μM
    • Follow PCR reaction mix as described in Table 2-1

    • Table 2-1: PCR Reaction Mix
      ComponentVolume (μL)Concentration (μM)
      5X Phusion Buffer101X
      10 mM dNTPs1200
      Primer A2.50.5
      Primer B2.50.5
      Template DNA~1-
      Phusion DNA Polymerase0.50.2 U/μM
      Nuclease-Free WaterFill to 50 μL-
      Total50-

      Note: Protocol will change for different enzymes
    • Use New England Biolabs' (NEB) Q5 2X Master Mix, perform at least 2 reactions so that you have plenty to work with in downstream applications
    • Pipette mixture up and down
    • Complete PCR Thermalcycler program as described in Table 2-2

    • Table 2-2: PCR Thermalcycler Program
      Cycle Step Temperature (°C) Time Cycles
      Initial Denaturation 98 30 s 1
      Denaturation
      Annealing
      Extension
      98
      Lowest Tm + 3
      72
      10 s
      15 s
      30 s/1 kbp
      30
      Final Extension 72 10 min 1
      Hold 4 - -


    • Run electrophoresis gel to check PCR product
  3. Use PCR clean-up kit to clean up PCR samples if successful
    • Store PCR product at 4 °C or -20 °C until further use
    • If PCR unsuccessful, refer to manual for further troubleshooting suggestions

Isolate the target plasmid

  1. Identify if your plasmid is high-copy or low-copy
    • For a high-copy plasmid, the miniprep from one 2 mL overnight culture should be sufficient
    • For a low-copy plasmid, minipreps from 2 to 4 mL overnight cultures should work
  2. Store plasmid at -20 °C until further use

Restriction Enzyme District

  1. If cutting with two enzymes identify if there is a buffer that will give good activity for both enzymes
    • If yes: proceed using that buffer
    • If no: first cut with one enzyme then purify with PCR clean-up kit; then cut with 2nd enzyme
    Refer to NEB Website for help in setting up an enzyme digest
  2. Cut both PCR product and backbone (plasmid) using Table 2-3 as a rough guide.

  3. Table 2-3: Enzyme Digest Mix
    Component Volume (μL)
    10X NEB Buffer 5
    BSA Add to 100 μg/mL if desired
    DNA 1 μg
    Restriction Enzyme 10 units
    Nuclease-Free Water Fill to 50 μL
    Total 50

  4. Incubate for at least 1 hour at temperature corresponding to incubation temperature for enzyme used.
  5. After restriction enzyme digest, use PCR clean-up kit to remove enzyme and buffer from both the PCR product and plasmid

Dephosphorylate Restriction-Enzyme-Cut Plasmid

  1. Combine components as outlined in Table 2-4

  2. Table 2-4: Dephosphorylation Mix
    Component Volume (μL)
    Molecular-Grade Water 39.5
    10X NEB Buffer 3 10
    Plasmid DNA 50 (volume from PCR clean-up)
    Alkaline Phosphotase (CIP) 0.5
    Total ~100

  3. Incubate at 37 °C for at least 1 hour
  4. Purify using PCR clean-up kit

Ligation

  1. Determine concentration of DNA of the insert and the plasmid (nanodrop or run a gel for a relative concentration)
    • In the ligation mix, make sure there is a total DNA concentration of 1 - 10 ng/μL
    • Usually use a ratio of 2:1 to 6:1 of insert to backbone (plasmid)
  2. Add components as in Table 2-5

  3. Table 2-5: Ligation Mix
    Component Volume (μL)
    T4 DNA Ligase Buffer 2
    DNA Insert varies
    DNA Backbone (Plasmid) varies
    T4 DNA Ligase 1
    Nuclease-Free Water Fill to 20 μL
    Total 20

  4. Incubate at room temperature for at least 1 hour
  5. Heat inactivate at 65 °C for 10 minutes and place on ice to cool
  6. Store at 4 °C

Transformation

  1. Thaw competent cells on ice. Leave in microcentrifuge tube. Set water bath to 43 °C and allow LB + antibiotics plates to come to room temperature
  2. Add 1 - 5 μL of ligation to cells
  3. Incubate on ice for 30 minutes
  4. Heat shock cells for 30 seconds at 42 °C without shaking
  5. Place on ice for 2 minutes
  6. In hood, add 250 μL of SOC media to the tube and cap tightly
  7. Spread 100 μL of a 1:10 dilution of the cells on an LB + antibiotics plate
  8. Incubate overnight at 37 °C
  9. Store remaining culture at 4 °C. If nothing grows or a few cells grow, you can plate the remaining the next day

Confirm Correct Construction of Plasmid

  1. Prepare an appropriate amount of 15 mL cell culture tubes with 2 mL LB + antibiotic
  2. Streak and prepare for incubation
    • Remove an LB + antibiotic plate from 4 °C
    • Label 1 to X # of colonies
    • From the overnight transformation plate, touch a sterile toothpick to a single colony, streak on the LB plate of the appropriate number, and place toothpick in culture tube. Repeat with other colonies
  3. Incubate at 37 °C and 225 rpm overnight
  4. Next Day:
  5. Isolate the plasmid by using the miniprep kit on the overnight cultures
    • If you have a large number of plasmids, check the uncut plasmids on a 0.7% agarose gel without the gene to serve as a control
  6. Check the plasmid by digesting with the appropriate restriction enzyme following the mixture given by Table 2-6

  7. Table 2-6: Confirmation Mix
    Component Volume (μL)
    10X NEB Buffer 2
    BSA Add to final concentration of 100 μg/mL if desired
    Plasmid DNA 5
    Restriction Enzyme 0.5
    Nuclease-Free Water Fill to 20 μL
    Total 20

  8. Incubate for at least 1 hour at the appropriate temperature for the enzyme
  9. Run the plasmid digest on a 1% agarose gel



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