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17/09/13
From 2013.igem.org
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Revision as of 10:37, 18 September 2013
Contents |
Results from previous day
- Background control plate had growth, which was not expected. This suggests that pSB1C3 backbone religated.
- It was also thought that the Tod genes used were not from the PCR fusion experiment.
- Same experiment done again, with a new set of Tod genes from a PCR fusion.
- For digestion of backbone, already digested backbone from 01/08/2013 was used, which was digested with EcoRI and PstI.
Incubation of bacteria from the plates of previous day for mini prep
- 12 single colonies were picked from each plate and put into 15ml of broth
- 36 tubes were left incubating overnight at 37C
Sending pGEM-T vector with Tod insert clones to sequencing by PNACL
Digestion of new fusion PCR Tod genes
- Digestion with EcorI-HF and PstI-HF using Cutsmart buffer
- 500ng of DNA was digested from the following Tod DNA concentrations:
| Sample | Concentration ng/ul | 260/280 | 260/230 |
| Tod X | 49.5 | 1.89 | 1.99 |
| Tod F | 33.9 | 1.98 | 1.68 |
| Tob B | 37.4 | 1.82 | 1.70 |
- Volumes for double digest:
| Sample | DNA | Buffer | Water | EcorI-HF | PstI-HF |
| Tod X | 10.1ul | 3ul | 15.9 | 0.5 | 0.5 |
| Tod F | 13.4ul | 3ul | 12.6 | 0.5 | 0.5 |
| Tob B | 14.7ul | 3ul | 11.3 | 0.5 | 0.5 |
- Incubation at 37C for 30min
- Heat kill at 80C for 20min
Ligation of Tod genes and pSB1C3 backbone
- For background control, just the pSB1C3 backbone was used
- For positive control, RFP plasmid from transformation efficiency kit provide by iGEM was used
- Quick stick ligase was used and a different protocol from previous day
- Bioline Quick Stick Ligase protocol:
- Combine the vector and the insert in the appropriate ratio to make up no more than 100ng of DNA
- Adjust volume to 14ul with ddH2O
- Add 1ul of QS ligase
- Add 5ul of 4x QS Buffer (vortex before use)
- Mix thoroughly before pipetting
- Incubate at room temperature for 5min
- The volumes for ligation:
| Sample | DNA ul | Vector(pSB1C3) ul | QS ligase ul | Buffer ul | H20 ul |
| Tod X | 0.9 | 2 | 1 | 5 | 5.1 |
| Tod F | 0.8 | 2 | 1 | 5 | 5.2 |
| Tob B | 1 | 2 | 1 | 5 | 5 |
| Positive control | 1 | 2 | 1 | 5 | 5 |
| Background control | 0 | 2 | 1 | 5 | 6 |
