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1. Single Digests: Performed today. Incubated digested samples for 2hrs @ 37C. Refer to table below. Gene 10x Buffer BSA Protein H20 DNA RE Promoter 5.0uL 0.5uL 40.4uL 2.1uL 2.0uL, SPE LAMBDAcI 5.0uL 0.5uL 29.5uL 15.0uL 1.0uL, XBA 1 LAMBDAcI 5.0uL 0.5uL 29.5uL 15.0uL 1.0uL, SPE Terminator 5.0uL 0.5uL 38.0uL 4.5uL 2.0uL, XBA 1

NOTE: RE = Restriction Enzyme

2. Ligation: Performed today. Heat inactivated enzyme @ 65C for 15 min to kill enzymes.Ligate samples. Incubate ligated samples @ 16C for 1hour. Refer to table below. Genes 10x Buffer H20 Base Vector Insert DNA Ligase LAMBDAcI/Terminator 2.0uL 10.7uL 2.0uL (Term.) 4.3uL (LAMBDAcI) 1.0uL Promoter/LAMBDAcI 2.0uL 10.7uL 2.0uL (Pro.) 4.3uL (LAMBDAcI) 1.0uL

3. Double Digests: Performed today. After digestion, incubate samples for 2hrs @37C. Heat inactivate restriction site enzymes EcorI and PstI in a water bath @ 65C for 15 minutes to denature the enzymes, thus stopping/controlling digestion. Place digested samples in -20C freezer overnight. Gene 10x Buffer BSA Protein H20 DNA RE 1 RE 2 Promoter/LAMBDAcI 5.0uL 0.5uL 22.5uL 20.0uL 1.0uL, EcorI 1.0uL, PstI LAMBDAcI/Terminator 5.0uL 0.5uL 20.0uL 20.0uL 1.0uL, EcorI 1.0uL, PstI Base Vector 5.0uL 0.5uL 15.0uL 15.0uL 1.0uL, EcorI 1.0uL, PstI

NOTE: RE = Restriction Enzyme

4. Make/Pour Ampicillin Plates

5. Order Primers for Part Sequencing

6. Purified Plasmid Prep: Purified MCherry, Promoter, Terminator and Base Vector because sequencing results were poor.

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