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. EcoR V-rPERK- SmaILoxp-tpA-loxp

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

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

6. 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

7. 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.

8. 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

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

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

12. 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