Team:Lethbridge/Notebook September
1
Ligation, PCR & Digest
Ligation of High and Low RAP Constructs into PSB1C3
| Volume (μL) | Part |
|---|---|
| 0.5 | GBlock |
| 20 | Phusion 5x HF buffer |
| 2 | Biobrick suffix 10nm |
| 2 | Biobrick prefix 10nm |
| 1 | phusion polymerase |
| 1 | 10nm dNTPs |
| 72.5 | dH20 |
Total volume: 100μL
PCR Amplification
| Tempertaure (°C) | Time (s) |
|---|---|
| 98 | 180 |
| 98 | 30 |
| 68.7 | 30 |
| 72 | 45 |
| 72 | 300 |
| 4 | Hold |
Cycles: 34
Digest of RAP constructs with EcoR1 and Pst1:
| Volume (μL) | Part |
|---|---|
| 12 | PCR product |
| 1 | EcoR1 |
| 1 | Pst1 |
| 2 | Cutsmart buffer 10x |
| 4 | dH20 |
Total Volume: 20μL
Digest of PSB1C3 with EcoR1 and Pst1:
| Volume (μL) | Part |
|---|---|
| 2 | PSB1C3 |
| 0.5 | EcoR1 |
| 0.5 | Pst1 |
| 1 | Cutsmart buffer 10x |
| 6 | dH20 |
Total Volume: 10μL
Ligation of low and high RBS RAP constructs into PSB1C3:
| Volume (μL) | Part |
|---|---|
| 1 | PSB1C3 digest |
| 3 | low/high 1 RBS constructs |
| 1 | Ligase 10x buffer |
| 0.5 | ligase |
| 4.5 | dH20 |
Total Volume: 10μL
Ligation of low and high RBS RAP constructs into PSB1C3:
| Volume (ul) | Part |
|---|---|
| 1 | PSB1C3 digest |
| 6 | low/high 2 RBS constructs |
| 1 | Ligase 10x buffer |
| 0.5 | ligase |
| 1.5 | dH20 |
Total Volume: 10μL
2
In Vitro Transcription
In Vitro Transcription of PCR Products at Varied Conditions
| Volume (μl) | Part |
|---|---|
| 10 | 5x TRAB |
| 5 | DTT 100nM |
| 6 | NTPs 25nM |
| 6 | NTPs 25nM |
| 2.5 | 100nM GMP |
| 1 | 0.5units/μL IPPase |
| 0.25 | 40units/μL RNase |
| 5 | PCR template DNA |
| 15.25 | milliQ H20 |
| 5 | T7 RNA polymerase |
Total Volume: 50μL
Incubate reactions at 37°C room temp for one hour overnnight.
DNase digest for 20 minutes.
In-vitro transcription time course at 16°C, to observe full length Tt aptazyme
Time points 0, 1, 5, 10, 20, 40, 80 minutes
| Volume (μL) | Part |
|---|---|
| 2 | 5x TRAB |
| 1 | DTT 100nM |
| 1.2 | NTPs 25nM |
| 0.5 | 100nM GMP |
| 0.2 | 0.5units/μL IPPase |
| 0.05 | 40units/μL RNase |
| 1 | PCR template DNA |
| 3.15 | milliQ H20 |
| 1 | T7 RNA polymerase |
6
Overexpression
Overexpression of RAP Low and High RBS-MS2
| Time Point (hrs) | Optic Density (OD) Low RBS | Optic Density (OD) High RBS |
|---|---|---|
| T1/2 | 0.8 | 1.08 |
| T1 | 1.2 | 1.46 |
| T2 | 1.5 | 1.89 |
| T3 (end) | 1.61 | 1.77 |
Gels
| Position | content |
|---|---|
| 1 | Tend Low |
| 2 | T2 Low |
| 3 | T1 Low |
| 4 | Ladder |
| 5 | T30 Low |
| 6 | T0 |
| Position | Content |
|---|---|
| 1 | Ladder |
| 2 | T0 |
| 3 | T30 High |
| 4 | T1 High |
| 5 | T2 High |
| 6 | Tend High |
| 7 | Ladder |
| 8 | T0 High |
8
SDS Page
| Position | content |
|---|---|
| 1 | PL |
| 2 | T0 Low |
| 3 | T30 Low |
| 4 | T1 Low |
| 5 | T2 Low |
| 6 | Tend Low |
| 7 | - |
| 8 | T0 High |
| 9 | - |
| 10 | T30 High |
| 11 | T1 High |
| 12 | T2 High |
| 13 | Tend High |
| 14 | PL |
9
Restriction Digest
Restriction Digest of PSB1C3 Tt Test RAP High:
| Volume (μL) | Part |
|---|---|
| 5 | PSB1C3 Tt Test RAP high |
| 1 | EcoR1 |
| 1 | Pst1 |
| 2 | Cutsmart Buffer 10x |
| 12 | dH20 |
Total Volume: 20μL
10
Miniprep
Miniprep of Tt Test Cultures 1-5:
- 3mL of culture centrifuged at 13,300rpm for 2 minutes; drain supernatent.
- Add 100μL solution 1, 200μL solution 2 and 350μL solution 3 in order.
- Centrifuge at 13,300rpm for 5 minutes; drain supernatent.
- Move supernatent to EZ tube; centrifuge at 10,000rpm for 2 minutes; drain supernatent.
- Add 750μL of wash solution, centrifuge at 10,000rpm for 2 minutes; drain supernatent. Repeat.
- Transfer column to 1.5mL tube, centrifuge at 10,000rpm for 2 minutes; drain supernatent.
- Elute with 50μL warm dH20, centrifuge at 10,000rpm for 2 minutes.
13
PCR
Diagnostic PCR of Tt Test and RAP High RBS
| Volume (μL) | Part |
|---|---|
| 5 | Quanta AccuStart Supermix 2x |
| 1 | Plasmid DNA colony suspension |
| 0.5 | BB prefix T7 promoter |
| 0.5 | BB suffix primer |
| 3 | dH20 |
Total Volume: 10μL