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Team:Groningen/Labwork/23 July 2013
From 2013.igem.org
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<br>Cut the DNA from the miniprep with EcoRI and PstI restriction enzymes, to check if the gene is there. | <br>Cut the DNA from the miniprep with EcoRI and PstI restriction enzymes, to check if the gene is there. | ||
<br>If all goes well to bands should be visual on gel on at 5191 bp and on at 1110 bp. | <br>If all goes well to bands should be visual on gel on at 5191 bp and on at 1110 bp. | ||
| - | <br>Tubes with DNA and enzymes were | + | <br>Tubes with DNA and enzymes were placed in the 37C incubator for 1.5 hours. A gel was run at 90V for 35 min. |
| - | + | ||
| - | + | ||
Revision as of 13:44, 23 July 2013
Mirjam
Run a gel with the PCR products of silk without strep + signal sequence.
The PCR of silk without strep + signal sequence is successful. A new 1.5% agarose gel is made to do gel purification. Did the gel purification. When checked over gel a faint band is seen for some of them. So the samples are combined and concentrated. Concentration is very low. So a PCR is made with the use of this PCR product.
Run a gel with the purified PCR product of silk strepF-R and silk F-strepR.
The products are present in 2/3 samples for each silk type. These are combined and concentrated. Concentrations of 18.1 and 16.1 ng/ul are obtained. So a restriction digestion can be made.
Made a restriction digestion for the signal sequences. These look fine when checked over gel.
Made a new restriction digestion for silk strepF-R and silk F-strepR. Run a gel to determine if the restriction is performed correctly.
Sander
Made a restriction digestion for silk strepF-R and silk F-strepR. No bands are seen on gel.
Inne
E. coli with the BBa_818000 backbone that had to grow overnight grew.
| Sample | Composition | BBa_k818000 from | growth |
| 1 | 4 mL LB, 8 uL Amp | iGEM 2012 | Yes |
| 2 | 4 mL LB, 8 uL Amp | iGEM 2012 | Yes |
| 3 | 4 mL, LB 8 uL Amp (negative control) | iGEM 2012 | No |
| 4 | 4 mL LB, 8 uL Amp, 4 uL IPTG | iGEM 2012 | Yes |
| 5 | 4 mL LB, 8 uL Amp, 4 uL IPTG | iGEM 2012 | Yes |
| 6 | 4 mL, 8uL Amp | iGEM 2013 | Yes |
| 7 | 4 mL, 8 uL Amp, 8uL IPTG | iGEM 2013 | Yes |
| 8 | 4 mL, 8 uL Amp (negative control) | iGEM 2013 | No |
All suspensions were white. Decided to do a miniprep + digestion to see is plasmid was present. Samples 1 and 6 were chosen, 1 to check if renewed 2012 inoculation was a success and 6 to check if our stock had a the backbone. Upon down spinning the cells (first step of miniprep) a pinkish color was detected in the pellet of cell sample 6. This suggest that the BBa_k818000 backbone was in there after all. Sample 1 showed a less conclusive result. The cells weren't pure white, but also didn't display the same level of pinkness as sample 6.
Fast Restriction digest:
Cut the DNA from the miniprep with EcoRI and PstI restriction enzymes, to check if the gene is there.
If all goes well to bands should be visual on gel on at 5191 bp and on at 1110 bp.
Tubes with DNA and enzymes were placed in the 37C incubator for 1.5 hours. A gel was run at 90V for 35 min.
