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Exeter/14 August 2013
From 2013.igem.org
MiniPrep
We Miniprepped the following parts:
- Promoter and RBS ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K608002 BBa_K608002])
- Magenta pigment ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K592012 BBa_K592012])
- Yellow pigment ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K592010 BBa_K592010])
- Yellow pigment with RBS ([http://parts.igem.org/wiki/index.php?title=Part:BBa_S05058 BBa_S05058])
- Terminator ([http://parts.igem.org/wiki/index.php?title=Part:BBa_B0015 BBa_B0015])
- RFP control ([http://parts.igem.org/wiki/index.php?title=Part:BBa_J04450 BBa_KJ04450])
NanoDrop results
| Part | ng/ul |
|---|---|
| RFP control | 80.7 |
| Yellow w/ RBS | 60.8 |
| Promoter and RBS | 127.8 |
| Magenta pigment | 46.9 |
| Terminator | 89.9 |
| Yellow pigment | 59.0 |
| KAN plasmid | 23.6 |
Digestion
| Part | ng/µl from NanoDrop | µl need to have 250ng DNA | 16µl - required µl DNA (amount of water needed for digestion) |
|---|---|---|---|
| RFP control | 80.7 | 3.10 | 12.9 |
| Yellow w/ RBS | 60.8 | 4.11 | 11.89 |
| Promoter and RBS | 127.8 | 2.0 | 14.0 |
| Magenta pigment | 46.9 | 5.33 | 10.67 |
| Terminator | 89.9 | 2.80 | 13.2 |
| Yellow pigment | 59.0 | 4.24 | 11.76 |
| KAN plasmid | 23.6 | 10.60 | 5.4 |
Part A's (magenta pigment, yellow pigment, yellow pigment with RBS and RFP control) cut with EcoRI and SpeI.
Part B (terminator) cut with XbaI and PstI.
KAN plasmid cut with EcoRI, PstI and DpnI.
Incubated at 37oC for 30 minutes, then 80oC for 20 minutes.
Gel of digest
| Lane | Part |
|---|---|
| 1 | empty (well looked damaged) |
| 2 | 1kb GeneRuler Ladde |
| 3 | Terminator |
| 4 | RFP control |
| 5 | skipped, looked damaged |
| 6 | skipped, looked damaged |
| 7 | Magenta pigment |
| 8 | KAN plasmid |
| 9 | Yellow pigment w/ RBS |
| 10 | Yellow pigment |
| 11 | 1kb GeneRuler Ladder |
Wells 5 and 6 are empty (as expected! There's no DNA in them!) however lane 8 is empty when we expect to see bands for the KAN plasmid. The KAN plasmid gave a reasonably low NanoDrop value; possibly there is insufficient DNA in the well to show bands. This would also be likely if the DNA has settled to the bottom of the Eppendorf the digest was set up in, and then the sample needed to run in the gel was pipetted from the top portion of the Eppendorf, where little DNA is present. This highlights the importance of our "vortex everything" rule!
Ligations
| Parts to Ligate | DNA(pigment)+DNA(term)+DNA(KAN plas) | <- equals | +T4 buffer (1ul) and ligase (0.5ul) | no-nuclease water needed (ul) |
|---|---|---|---|---|
| Magenta to Terminator | 0.648+0.12+2.0 | 2.768 | 4.268 | 5.732 |
| Yellow w/ RBS to Terminator | 0.680+0.12+2.0 | 2.800 | 4.300 | 5.700 |
| Yellow to Terminator | 0.664+0.12+2.0 | 2.784 | 4.284 | 5.716 |
| RFP control | 0.992+0.12+2.0 | 3.112 | 4.612 | 5.388 |
Ligated at 16oC for 30 mins, then 80oC for 20 mins. Transformed into DH5α competent cells.
Take me back to the notebook.
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