Exeter/14 August 2013

From 2013.igem.org

Exeter iGEM 2013 · Paint by Coli


We Miniprepped the following parts:

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


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
Image: 500 pixels

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!


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.

Exeter iGEM 2013 · Paint by Coli