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Exeter/1 August 2013
From 2013.igem.org
Please not that EcoRI, XbaI, PstI and SpeI are refereed to as "E, X, P and S" respectively throughout this page.
Miniprep
[http://parts.igem.org/Part:BBa_B0015 B0015]
[http://parts.igem.org/Part:BBa_K592018 K592018]
[http://parts.igem.org/Part:BBa_S05058 S05058]
Nanodrop
| Culture | Nanodrop concentration (ng/ul) | for digest (ul) |
|---|---|---|
| B0015 | 30.9 | 8.1 |
| K592018 | 106.2 | 2.4 |
| S05058 | 58.2 | 4.3 |
| RFP | - | - |
Nanodrop of digests
We are worried that there is insufficient DNA in our restriction digests, as our gels have no bands.
Group 1
Eluted with purite water, digestion by Adam using Victoria's protocol.
1 - RBS + Cph8 (E+S) - 43.9 ng/ul
2 - B0015 (x+p) - 26.7 ng/ul
3 - Negative control - 23.3 ng/ul (gives a reading due to NEB2 and BSA)
4 - Positive control (E+S) - 37.3
5 - Positive control (X+P) - 18.8
Group 2
Eluted with RNAse-free water (But possibly contaminated)! Digestion by Rachel and Flick using Victoria's recipe.
A - RBS + Cph8 (E+X) - 89.3
B - RBS + cyan (X+E) - 63.5
C - B0015 (E+S) - 66.0
D - AMP plasmid (E+P+D) - 40.4
E - Positive control (E+S) - 51.5
F - Positive control (X+P) - 57.3
G - Negative control (E+S) - 33.9 (gives a reading due to NEB2 and BSA)
Third retry of digestion
We have new no-nuclease water from Paul.
Instead of using an RFP plasmid from a transformation/mini-prep, we're using one resuspended from a kit plate.
The reason we're not seeing anything on the gels may be because we're not adding enough DNA.
We've split into 2 teams : Clio and Victoria Recipes:
Victoria's Recipe
| Culture | no-nucleotide water | DNA | NEB2 | BSA | E | X | S | P | |
|---|---|---|---|---|---|---|---|---|---|
| 1. RBS + Cph8 | 5.0 | 10 | 2.5 | 0.5 | 1.0 | 1.0 | - | - | |
| 2. RBS + cyan | 5.0 | 10 | 2,5 | 0.5 | 1.0 | 1.0 | - | - | |
| 3. B0015 | 5.0 | 10 | 2.5 | 0.5 | 1.0 | - | 1.0 | - | |
| 5. Positive control (E+S) | 5.0 | 7 | 3 | - | 1.0 | - | 1.0 | - | |
| 7. Negative control | 15.0 | - | 3 | - | 1.0 | - | 1.0 | - |
37°C for 10mins, 80°C for 20mins.
Clio's Recipe
| Culture | water | DNA | 10x fast buffer w/green | E | X | S | P | D | |
|---|---|---|---|---|---|---|---|---|---|
| A. RBS + Cph8 | 3 | 10 | 5 | 1.0 | 1.0 | - | - | - | |
| B. RBS + cyan | 3 | 10 | 5 | 1.0 | 1.0 | - | - | - | |
| C. B0015 | 3 | 10 | 5 | 1.0 | - | 1.0 | - | - | |
| D. AMP plasmid | 3 | 7 | 5 | 1.0 | - | - | 1.0 | 1.0 | |
| E. Positive control RFP (X+P) | 5 | 10 | 5 | - | 1.0 | - | 1.0 | - | |
| G. Negative control | 13 | - | 5 | 1.0 | - | 1.0 | - | - |
37°C for 30mins , 80°C for 20mins.
We didnt have much resuspended RFP (hence 7ul DNA used) so 1 each. We also didt have enough AMP plasmid to do one each.
Gel
Lane 1 - 1kb GeneRuler ladder
Lane 2 - 1. RBS + Cph8
Lane 3 - 2. RBS + Cyan
Lane 4 - 3. B0015
Lane 5 - 5. RFP control E+S
Lane 6 - 7. negative control
Lane 7 - A. RBS + Cph8
Lane 8 - B. RBS + cyan
Lane 9 - C. B0015
Lane 10 - D. AMP plasmid
Lane 11 - E. RFP control X+P
Lane 12 - G. Negative control
Lane 13 - 100bp plus DNA ladder.
Neither worked! No DNA visible.
Take me back to the notebook.
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