Team:Evry/Interlab Study/08-14-2014

Contructions PSB1C3 with I20260, J23101-E1010 and K823012-E1010

To amplify BBa_I20260 and BBa_0240, a PCR was perform with the mix described Table 1. The program IGEM Q5 PCR was applied, see Table 2. PCR products were cleaned with the GeneJET purification kit (Thermo Scientific)followed by DNA quantification with the NanoDrop 2000 (Thermo Scientific). Purified samples of I20260 and E1010 had respectively following concentration 30.9 ng/µl and 43.3 ng/µl.

Preparation of a 1% agarose gel: 0.52 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X. Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 20 µl of BBa_E0240 PCR product and BBa_I20260 purified PCR product added with 4 µl of loading dye, at 10µl of the mix per well. Gel running 45 minutes at 100 mV in TAE 1X buffer.

Figure 4: 1% agarose gel of PCR products from 08.14.2014 after PCR clean up. Lane 1 and 5: Purple 2-Log ladder NEB, Lane 2, 3 and 4: BBa_E0240 purified PCR product, Lane 6,7 and 8: BBa_I20260 purified PCR product

3 pieces of gel were sampled in four tubes to perform a DNA purification from gel. The DNA purification was made with the GeneJET purification kit (Thermo Scientific).
A verification electrophoresis was performed on a 1% agarose gel.

Figure 5: 1% agarose gel of purified fragments from gel. Lane 1: Purple 2-Log ladder NEB, Lane 2: BBa_E0240 1200 bp purified fragment and Lane 3: BBa_I20260 1200 bp purified fragment

The major part of the DNA was lost during the purification because the amount of was to weak to be purified from gel. So we decide do transform ''E. coli'' to isolate a colony containing the desired plasmid for BBa_E0240 and BBa_I20260.