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Team:University of Ottawa/30 July 2008
From 2008.igem.org
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:<li>T123 amplification product was run on a gel and extracted. | :<li>T123 amplification product was run on a gel and extracted. | ||
:<li>24 gel extraction were performed in total, care was taken to minimize UV exposure (samples were not exposed for longer than 20s) | :<li>24 gel extraction were performed in total, care was taken to minimize UV exposure (samples were not exposed for longer than 20s) | ||
| + | '''Gel of extracted PCR amplification product''' | ||
| + | :<li> Expected bands were seen, the T123 is good to go. | ||
| + | '''0A and 0B absorbances''' | ||
| + | :<li> absorbances of 0A and 0B indicated very good concentrations | ||
| + | '''Ligation and digestion''' | ||
| + | :<li> Digestion (with SphI and PstI) of T123 0A and 0B were all performed and left overnight | ||
Latest revision as of 14:57, 12 August 2008
Contents |
Today in the Lab
Tammy
| Sample | Concentration (ng/μL) | V (μL) | Total DNA (ng) |
| pSSA14 | 129 | 7 | 903 |
| OB | 170 | 6 | 1020 |
- Digestion Step 1 of pSSA14 with PstI
| Digestion Reaction components | Vol (μl) |
| H2O | 4 |
| Buffer 3 | 1.5 |
| BSA | 1.5 |
| PstI | 1 |
| DNA template | 7 |
| Total | 15 |
- Digestion Step 1 of OB with PstI
| Digestion Reaction components | Vol (μl) |
| H2O | 5 |
| Buffer 3 | 1.5 |
| BSA | 1.5 |
| PstI | 1 |
| DNA template | 6 |
| Total | 15 |
- Digestion Step 2 of pSSA14 with ClaI
| Digestion Reaction components | Vol (μl) |
| H2O | 9.5 |
| Buffer 4 | 3 |
| BSA | 1.5 |
| ClaI | 1 |
| DNA template | 15 |
| Total | 30 |
- Digestion Step 2 of OB with ClaI
| Digestion Reaction components | Vol (μl) |
| H2O | 9.5 |
| Buffer 4 | 3 |
| BSA | 1.5 |
| ClaI | 1 |
| DNA template | 15 |
| Total | 30 |
Chris
PCR Confirmation
- Ran all 5 PCR product samples on a 1% gel for 40 minutes at 80 V
- Desired band appeared on gel; PCR amplification was successful.
- Used PCR cleanup kit to purify samples
- Measured absorbance of PTP2 samples
Digestion of PTP2
- Digested PTP2 with BamHI and XhoI; ran 5 tubes in parallel. Incubated at 37 C for on hour. Ran one water control.
- Used PCR cleanup kit to purify digestion products. Measured absorbance to determine concentration.
- Concentrations too low for ligation.
Matt
- Measured absorbance of pSSA42 gel extraction products from digestion.
Dan
Gel Extraction
- T123 amplification product was run on a gel and extracted.
- 24 gel extraction were performed in total, care was taken to minimize UV exposure (samples were not exposed for longer than 20s)
Gel of extracted PCR amplification product
- Expected bands were seen, the T123 is good to go.
0A and 0B absorbances
- absorbances of 0A and 0B indicated very good concentrations
Ligation and digestion
- Digestion (with SphI and PstI) of T123 0A and 0B were all performed and left overnight
