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EcoR V -r PERK - SmaI

From rPERK to myc-tagged mPERK: Construction of pShuttle-loxp-tpA-loxp-mPERK_9E10-IRES-hrGFP-1 plasmid for conditional rescued PERK KO mouse. Loxp- tpA -loxp. EcoR V -r PERK - SmaI. A myc-tagged mouse PERK became available. Better to use mouse PERK in mouse system

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EcoR V -r PERK - SmaI

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  1. From rPERK to myc-tagged mPERK:Construction of pShuttle-loxp-tpA-loxp-mPERK_9E10-IRES-hrGFP-1 plasmid for conditional rescued PERK KO mouse

  2. Loxp-tpA-loxp EcoR V-rPERK- SmaI

  3. A myc-tagged mouse PERK became available • Better to use mouse PERK in mouse system • No good antibody available for PERK

  4. Mysterious Plasmid Degradation • Happened first time on pcDNA3.1+ extracted from transfected electro-competent cells • (Digested with EcoR I, Nhe I and XhoI)

  5. DNase Contamination? • Extraction Kit? • No problem with other plasmids extracted • Something Wrong with Enzymes? • Degradation happens even without enzymes • Buffers contaminated? • Fine for other plasmids • Problem disappeared after the previous kit was used up and the new one came in

  6. Mysterious Plasmid Degradation Once More • Ligation products of mPerk_9E10 and p-shuttle-loxp-tpa-loxp fragments extracted from transfected electro-competent cells get degraded in buffers.

  7. Really Strong DNase • Boiling of DNA for 5 min • Phenol-Chloroform Extraction and Alcohol reprecipitation • Traditional alkaline lyses extraction None of them worked • Degrades other good plasmids when incubated together in enzyme buffer

  8. Strain problem? Transfect them into DMSO competent cells from JW.

  9. Digest with Hind III • Expected fragments : 8690, 2608, 1127, 894, 220 • Control (psh-tpa-rPERK) 8960, 3249, 1385

  10. Future Work • Transfect into regular cell line and TE-CRE cell line to confirm CRE-controlled mPERK_9E10 expression • DNA level: PCR detection of shortened version of plasmid • RNA level: RT-PCR to detect myc-tag expression • Protein level: western-blot with myc-tag antibody • Functional level: More eIF2alpha phosphorylation upon ER stress

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