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From 2013.igem.org

Contents 1 Isolation of ligated plasmid 2 Digestion of ligated plasmid 3 Gel Electrophoresis 4 Single digest

Isolation of ligated plasmid

• The ligated plasmid- chloramphenicol backbone and limonene biobrick, were isolated from cells that were incubated overnight in broth by miniprep.
• Protocols from both Bioline kit and Thermoscientific were used for the miniprep.
• there were 6 tubes containing the plasmid.
• The plasmid concentration in each of the 6 tubes were measured using nanodrop.
• 200ng of plasmid from each of the 6 different tube was used for digestion.

sample	volume(ul)	concentration (ng/ul)	yield(ug)
5.1	99	75	7.4
5.2	100	44.2	4.4
5.3	98	46.7	4.6
10.1	46	61.8	2.84
10.2	44	77.1	3.39
10.3	47	13.7	0.64

Digestion of ligated plasmid

• Master Mix (changes were made from the protocol)
  • 2ul of NEB Buffer
  • 1ul of EcoRI
  • 1ul of PstI
  • varying concentration of dH2O and plasmid depending on overall concentration of plasmid in each tube.
  • Final volume of each of the 6 tubes was 20ul.

Gel Electrophoresis

• The product of digestion was run on agarose gel
  • This was done in order to confirm the presence of the limonene biobrick

• The lanes are in the following order:
  • Marker, Sample 5.1, Sample 5.2, Sample 5.3, Sample 10.1, Sample 10.2, Sample 10.3, Marker.
• We were expecting to see a 2kb and 5kb band. These bands appeared for samples 5.1, 10.1 and 10.2. To further determine the composition of samples, we did a single digest on the samples.

Single digest

• For samples 5.1, 10.1, 10.2 and 10.3 two single digests each were made with EcoRI and PstI, giving a total of 8 samples.
• The samples are run on 0.8% agarose gel.