User:Matt Hartings/Notebook/Photosynthesis/2013/01/04

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Objective

Prepping some work for the weeks ahead

Cloning

PCR

  1. I'm going to try to do the Mb cloning into pQE-80 myself. (May want to try pDrive Coning Vector eventually)
    1. The following protocol is taken from here
    2. PCR mix
      1. 10X reaction Buffer
        1. 2.5μL
      2. NTP's stock solution is 10mM (make 50μL of 2mM by diluting 10μL of stock)
        1. 2.5μL
      3. 3' primer (25μM) (need to check my concentrations for rough estimates)
        1. 1.0μL
      4. 5' primer (25μM) (need to check my concentrations for rough estimates)
        1. 1.0μL
      5. DNA template (0.1μg/μL) (need to check my concentration for rough estimate)
        1. 1.0μL
      6. Autoclaved water
        1. 16.5μL
      7. Pfu polymerase
        1. 0.5μL
    3. PCR cycles
      1. 1' 94°C
      2. 1' 55°C
      3. 1' 72°C
      4. Repeat previous three steps 3 times
      5. 1' 94°C
      6. 1' 60°C
      7. 1' 72°C
      8. Repeat previous three steps 12 times
      9. 7' 72°C

Purifying PCR product

  1. Run a 1% Gel
    1. (25 mL TBE, 0.25g agarose)
  2. Incubate gel in ethidium bromide solution for 30 minutes
  3. Incubate gel in rinse solution for 30 minutes
  4. Look for band on illuminator
    1. If band exists, cut out of gel with razor
  5. Follow protocol from Promega Wizard miniprep kit
    1. melt the gel for 5-10' at 70°C
    2. add 1mL purification resin and mix
    3. put the resin in the column
    4. wash with 2mL 80% isopropanol
    5. spin 2' at 14000 rpm
    6. air dry for 5'
    7. add 50uL autoclaved water and incubate 2'
    8. spin 30 seconds at 14000 rpm

Restriction of PCR product

If you had a bright DNA band after PCR, use 20μL of the product. If you had a faint band, lyophilize the 50μL from the previous step and add 20μL water. The restriction enzymes that I need for pQE-80 are: BamHI and HindIII

  1. Start protocol first thing in the morning
  2. Do the restriction in a total 50μL mix
    1. normally choose the buffer that is good for both enzymes, but NEB says to do them sequentially
  3. Add 5μL HindIII buffer
  4. Add 24μL water
  5. Add 1μL HindIII
  6. Incubate for day at 37
  7. Add 1μL of 5M NaCl to buffer see discussion thread
  8. Add 1μL BamHI
  9. Incubate overnight
  10. Heat inactivate 20' at 65°C
  11. (Don't know if I need to gel purify here)

Restriction of vector

Run the cuts at the end of the day

  1. cut 10μg vector in a 50μL mix
    1. Add appropriate volume of DNA (check concentration)
    2. Add 5μL HindIII buffer
    3. Add appropriate volume of water to 49μL
    4. Add 1μL HindIII
    5. Incubate at 37°C overnight
  2. Run a 1% Gel
    1. (25 mL TBE, 0.25g agarose)
  3. Incubate gel in ethidium bromide solution for 30 minutes
  4. Incubate gel in rinse solution for 30 minutes
  5. Look for band on illuminator
    1. If band exists, cut out of gel with razor
  6. Follow protocol from Promega Wizard miniprep kit
    1. melt the gel for 5-10' at 70°C
    2. add 1mL purification resin and mix
    3. put the resin in the column
    4. wash with 2mL 80% isopropanol
    5. spin 2' at 14000 rpm
    6. air dry for 5'
    7. add 50uL autoclaved water and incubate 2'
    8. spin 30 seconds at 14000 rpm
  7. Lyophilize and resuspend in 20μL water
  8. perform second cut on vector in a 50μL mix
    1. Use 20μL from previous gel purification
    2. Add 5μL BamHI buffer
    3. Add 24μL water
    4. Add 1μL BamHI
    5. Incubate at 37°C overnight
  9. Run a 1% Gel
    1. (25 mL TBE, 0.25g agarose)
  10. Incubate gel in ethidium bromide solution for 30 minutes
  11. Incubate gel in rinse solution for 30 minutes
  12. Look for band on illuminator
    1. If band exists, cut out of gel with razor
  13. Follow protocol from Promega Wizard miniprep kit
    1. melt the gel for 5-10' at 70°C
    2. add 1mL purification resin and mix
    3. put the resin in the column
    4. wash with 2mL 80% isopropanol
    5. spin 2' at 14000 rpm
    6. air dry for 5'
    7. add 50uL autoclaved water and incubate 2'
    8. spin 30 seconds at 14000 rpm

Ligation

This also runs overnight

  1. Premix vector insert and Mg2+
    1. 1μL cut vector
    2. 5μL insert
    3. 1μL Mg2+ (20x)
    4. 5μL H2O
  2. Incubate mix for 5' at 37°C
  3. Add 2μL 10x ATP/DTT (10mM/100mM)
  4. Add 1μL T4 DNA ligase
  5. Incubate 2 hours at RT and then ON at 16°C

Transform

Protocol uses electrocompetent cells. May have to see about using these


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