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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