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

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Week 2 Day 3


- 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.