IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2014/06/25

iGEM Project name 1

Main project page Previous entry      Next entry

Week 2 Day 3 TSL - Midi prep following the Qiagen protocol of the 9 constructs previously synthesised by DNA 2.0 and transformed on Day 2 and DNA conc. of each constrcut determined using the nanodrop. Dig-Lig calculation spread sheet completed using these concentrations. - Set up the first 11 Dig-Lig experiments, placed into PCR block on the slow protocol. - Carbenicillin plates made and spreadplated with 40 μl of XGAL and 100 μl of IPTG. Plates left to dry upside down in 37°C incubator. - Transformation of GG DNA into electrocompetent E.coli cells, PROTOCOL: • Cells removed from -80°C freezer and allowed to thaw. • A 5 µl sample of DNA was added to a 50 µl aliquot of cells. • Transfered each 55 µl sample into a cuvette. • Electroporation pulse on setting ECR1. • A 500 µl of SOC broth was added to the cuvette. • A 555 µl cuvette sample was transferred to an eppendorf tube and incubated for 40 min at 37°C. • A 100 µl sample was spreadplated onto agar plates containing carbenicillin and incubated O/N at 37°C. - A 20 µl PCR reaction was set up using a GG level 1 acceptor as template DNA containing the primers P1: 0349(PRO+5U) and 0353 (3U+TER) and P2: 0350 (PRO+5U) and 0354 (3U+TER). - A TBE buffer 1% agarose gel was poured for diagnostic purposes to check the amplification of the PCR products. - PCR product purification • Pre-percipitation to remove TAQ polymerase and protein was completed followed by phenolchloroform extraction (x2). Aqueous layer is removed and discarded leaving DNA. • Precipitation to remove buffer was carried out by adding Na acetate (10%) and ethanol causing the DNA to come out of solution. Aspirator was used to remove buffer, salt and ethanol. • A salt wash was completed to remove the acetate and ethanol. • Sample was incubated at 65°C for 2 min to evaporate the ethanol.