iGEM 2014 Measurement Interlab

1. First we transformed E. coli Dh5alpha according to iGEM protocol with the following parts from iGEM distribution kit 2014:
• BBa_I20260 (Plate 4, Well 18A)
• BBa_J23101 (called BBa_K823005 when in pSB1C3): Plate 1, Well 20K
• BBa_E0240 (in pSB1C3): Plate 2, Well 24B
• BBa_J23115 (called BBa_K823012 when in pSB1C3): Plate 1, Well 22I
• BBa_E0240 (in pSB1C3): Plate 2, Well 24B

2. After we observed the growth of transformed bacteria on chloramphenicol selective plates, each part was extracted using PureLink® HiPure Plasmid Miniprep Kit (Invitrogen, catalog number K2100-02). The concentration of the plasmids was checked with Nanodrop.

PCR of the parts was done according to the following protocol for 20ul reaction (adapted from http://www.thermoscientificbio.com/uploadedFiles/Resources/tech-manual-f-531-f-532-phusion-high-fidelity-pcr-master-mix.pdf):
• Sterile water – 4ul, high fidelity master mix – 10ul, VF2 primer – 2ul, VR primer – 2ul, DNA from miniprep (final concentration in the reaction=0.2ng/ul) – 2ul.
• Thermocycler program for the parts was adapted from http://www.thermoscientificbio.com/uploadedFiles/Resources/tech-manual-f-531-f-532-phusion-high-fidelity-pcr-master-mix.pdf and the extension time was adjusted according to the size of the part (25s per 1000bp).

Figure 1. Agarose gel electrophoresis of PCR products: first lane – 500bp gene ladder, second and third lanes – PCR products of Anderson promoters for Interlab study (35bp each)

Figure 2. Agarose gel electrophoresis of PCR product of GFP generator for Interlab study: 1st lane – 1kb gene ruler, 2nd lane – 876bp GFP generator amplified with PCR.