User:Daniel B Rosen/Notebook/Methylation Sensor/2011/10/01

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Daniel B Rosen 18:33, 1 October 2011 (EDT)

Cloning of pCR2-mCherry into 4 Plasmid vectors

Vectors

  1. pOriEBNA(CMV-ppGFP)
  2. pOriEBNA(EF1a-ppGFP)
  3. pCMV-ppGFP
  4. pEF1a-ppGFP

Rationale: mOrange was found to have too narrow an excitation-emission spectrum (563, 572..?)

Cloning Procedure

  1. Digest plasmid backbone
    1. Create master mix of EcoRI digest (x1, (x4))
      1. 3 uL EcoRI buffer (12)
      2. 0.3 BSA (1.2)
      3. 1 uL EcoRI (4)
      4. 24.7 uL H2O mq (98.8)
    2. Add 29 uL to 4 epindorf tubes
    3. Add 1 uL of plasmid, vortex, spin and incubate for 2.5 hours
  2. Digest PCRII-mCherry
    1. Digest mix
      1. 10 uL mCherry plasmid
      2. 3 uL EcoRI buffer
      3. 1 uL EcoRI
      4. 0.3 uL BSA
      5. 15.7 uL H2O
    2. vortex, spin and incubate for 2.5 hours
  3. Enzyme Inactivation
    1. Incubate 20 min at 65 C
  4. Alkaline Phosphatase Preparation (only the vector or the insert); usually whichever one is larger
    1. Add Preparation
      1. 3 uL Anarctice phosphatase buffer
      2. 1 uL Anctartic phosphatase

b. 15 min at 37 C c. 5 min at 65 C to inactivate 5. Gel: .5 g agarose/50mL TAE buffer. 5 uL Ethidium Bromide/100 mL gel. Prepared 150 mL gel Bands: l kb ladder| pCRII-mCherry| 1| Cherry | 2 | Cherry | 3 | Cherry | 4 6. Clean Up 1. place vector and insert cut out into a 1.5 mL eppendorf tube 2. add 3x L3 buffer 3. Place in 50C heat block, <= 10 min and then 5 min more after it dissolves 4. Load onto quick gel extraction column inside a wash tube 5. Centrifuge >12,000 g 1 min 6. 2-3 min additional spin, discard wash tube 7. add 50 uL elution buffer (incubate 1 min) 8. Elute into recovery tube, Spin 1 minute 12,000 g


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