Agarose gel electrophoresis. Agarose gel electrophoresis was used to check PCR reaction products and for visualisation of products of restriction. High resolution agarose was used for some genotyping. Gel was melted and poured into the gel tray with a well comb and allowed to set over 1 hour. 5ul PCR products were mixed with 2ul agarose loading buffer and the mixture loaded into 10ul xxxxx in 1.5% agarose gel in 1xTBE running buffer. 1ul marker ladder (usually ΦX174 HaeIII fragments) was loaded. Electrophoresis was at 80V for 40 minutes. The gel was then soaked in 10ug/ml ethidium bromide for 20 minutes followed by two 15 minute washes in water. Visualisation was under UVA light.
Agarose gel electrophoresis. Agarose gel was prepared with 0.8 g agarose (Sigma, UK) dissolved in 100 mL of Tris-base, acetic acid and EDTA (TAE) buffer (pH 8) (Thermo Xxxxxx, UK) by heating for 5 min in the microwave at medium heat. 10 µL of nucleic stain Gel Red™ (Biotium, USA) was added to the molten agarose and the mixture was left to set in UV-transparent gel tray (Bio Rad Laboratories, USA). 10 µL of each sample prepared in the previous section was mixed with 2 µL of concentrated (6x) DNA loading dye ( 30% (v/v) glycerol, 0.25% (w/v) bromophenol blue and 0.25% (w/v) xylene cyanol [139]. The mixture was then transferred into the xxxxx of the agarose gel and electrophoresis was performed at 100 V for 25 min in TAE buffer at room temperature.
Agarose gel electrophoresis. Agarose gels were prepared by adding powdered agarose to distilled water, usually 2% (w/v), and heating for 1min in the microwave. Once melted, 5μl ethidium bromide was added per 100mls, and a gel was poured and left at room temperature to solidify. Samples were prepared with loading buffer (1:5 ratio) and added to xxxxx. Gels were run at 100V until an adequate separation had been achieved and the relevant band were excised using a scalpel and a flat UV lamp.
Agarose gel electrophoresis. 2.8.3.1 One-dimensional agarose gel electrophoresis For general molecular biology applications, 1% agarose gels were prepared with TAE and samples were mixed with 1/6 sample volume of 6x DNA Gel Loading Dye (NEB). Mini gels (8 x 10 cm) were run in TAE buffer for 1 hour at 80 V (8 V/cm) in a commercially available electrophoresis apparatus from Bio-Rad. For visualising micrococcal nuclease digests (as described in 2.13.2), samples were separated through 1.3% agarose gels (8 x 10 cm) in TAE buffer for approximately 1 hour at 100 V. For analysis of in vitro DNA replication assays, 0.6%-2% agarose gels (15 x 10 cm) were run for 16 hours at 24 V under native (TAE) or denaturing conditions (30 nM NaOH and 2 mM EDTA). Following electrophoresis, denaturing gels were immediately fixed in 5% TCA for 40 minutes (with gentle rotation) at 4°C. Native gels were stained with EtBr as described in 2.8.4 prior to fixation with 5% TCA. Gels were then dried onto 3MM CHR paper (GE) and exposed as described in 2.8.5.
Agarose gel electrophoresis. Agarose gel electrophoresis was used for separation of DNA fragments by size. The constant mass to charge ratio of DNA molecules allows separation of linearized DNA fragments according to fragment size. Migration rates are dependent on the pore size of the gel, which in turn is determined by the agarose concentration. The DNA fragments together with the 1kb DNA ladder (NEB, USA) were prepared accordingly by adding 1X gel loading dye (NEB, USA) and were loaded carefully into the xxxxx. The gel was run at 100V for approximately 1 hour. The gel was visualised using a UV light device and the DNA fragments were isolated using a sterile razor blade and were placed at 4˚C until further processing. Excised bands were purified using a gel extraction kit (Qiagen) following manufacturers instructions.
