Team:DTU Denmark/notebookredoxilator

Construction of biobricks: The restriction products were evaluated on a 1% agarose gel (see figure 2).

Figure 2: Restriction analysis of the three biobricks.

July 19th

Construction of biobricks: The three plasmids were purified according to the protocol (link to protocol). Following the purification of the plasmids, a restriction analysis was performed using SpeI enzyme (10U) and incubated at 37ºC overnight.

Construction of biobricks: Some colonies were picked from the transformation plates and made overnight cultures in liquid media.

Construction of biobricks: Following purification, all of the PCR product was digested with XbaI (60U) and SpeI (30U). The mixtures were incubated at 37ºC. After 8 hours of incubation, more enzyme was added (XbaI (20 U) and SpeI (10U)) to ensure maximum digestion. Following enzymatic purification a gel was run to estimate the concentration of the vector and the PCR product (see figure 1).

Figure 1: Electrophoresis gel picture of the PCR products of the three biobricks and the vector. The band 11P at 1500 bp corresponds to the vector; the three other bands at 537 bp, 207 bp and 125 bp correspond to the fragments Cln, GFP and Cln_GFP respectively.

To meet the 3:1 ratio of fragment to vector 12μl fragment was mixed with 2μl vector, 2μl ligase buffer (10x), 1μl ligase, 3 μl Mq H20. The ligation of the fragment and the vector was carried out on PCR machine using a 3:1 ratio for 1.5 hours using the idea by Lund, A. H., M. Duch, and F. S. Pedersen. 1996. Increased cloning efficiency by temperature-cycle ligation. Nucleic Acids Res. 24:800-801. doi:l50400 [pii]. The final product was finally transformed into competent E coli cells.

Construction of biobricks: The 357bp Cln2 fragment was amplified by PCR using the primers BGHA351/BGHA362 and Phusion polymerase. Primers BGHA368/369 were used to amplify the GFP fragment and finally, primers BGHA368/353 were used to amplify the fragment containing the GFP fused with the Cln. Following amplification the PCR products were run on a 1% agarose gel and purified The primers added tails to the PCR product containing an XbaI site in the forward primer and a SpeI in the reverse primer to meet the “Silver fusion” standard BBF RFC 23.

Construction of biobricks:

Construction of biobricks:

Construction of biobricks: