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 pOriEBNA(CMV-ppGFP) pOriEBNA(EF1a-ppGFP) pCMV-ppGFP pEF1a-ppGFP Rationale: mOrange was found to have too narrow an excitation-emission spectrum (563, 572..?) Cloning Procedure Digest plasmid backbone Create master mix of EcoRI digest (x1, (x4)) 3 uL EcoRI buffer (12) 0.3 BSA (1.2) 1 uL EcoRI (4) 24.7 uL H2O mq (98.8) Add 29 uL to 4 epindorf tubes Add 1 uL of plasmid, vortex, spin and incubate for 2.5 hours Digest PCRII-mCherry Digest mix 10 uL mCherry plasmid 3 uL EcoRI buffer 1 uL EcoRI 0.3 uL BSA 15.7 uL H2O vortex, spin and incubate for 2.5 hours Enzyme Inactivation Incubate 20 min at 65 C Alkaline Phosphatase Preparation (only the vector or the insert); usually whichever one is larger Add Preparation 3 uL Anarctice phosphatase buffer 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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