Desarrollo demográfico en época de bonanza, 1996-2007.

Agarose gel electrophoresis

2.2.4.1. Separation of PCR products in agarose gels

2g agarose in 100ml 1 x TBE was boiled in the microwave. On cooling,

5)liI of ethidium bromide (10mg/ml) was added and the gel poured in to a

BRL midi gel tray. After setting, lOpI of PCR product was diluted with 5pl of loading buffer, loaded onto the gel and the gel electrophoresed at 1 0 0

volts for 20-40 minutes. The gel was transilluminated and photographed under ultraviolet (UV) light. The size of the amplified product was compared to a 100 base pair ladder loaded at the same time as the PCR product.

PCR products which had been digested by restriction enzymes were separated on 3% Nusieve/1% agarose gels.

2.2 4.2. Separation of restriction enzyme fragments of human DNA for Southern blotting.

2.4g agarose in 300ml 1 x TAB buffer was boiled in the microwave. On cooling, ISpI of ethidium bromide (lOmg/ml) was added and the gel poured in to a 20cm x 20cm NBL tray. Once set, lOpI loading buffer was added to each 40pl digest and the total volume loaded into a well. Molecular weight marker, Ipg of lambda DNA digested with BstE II, was

also loaded. Gels were electrophoresed at 50 volts for 16 hours, and photographed under UV light next to a fluorescent ruler.

Polyacrylamide gel electrophoresis. 2.2.4.3. Microsatellite analysis.

^^P-labelled dinucleotide repeat polymorphisms.

A pair of BRL sequencing plates were washed in detergent, rinsed with water, dried and wiped with 70% ethanol. The smaller plate was coated in Sigmacote (Sigma). Two 0.4mm spacers were placed along the edges of the two opposed plates and these were then clipped together using bulldog clips. Gel mix consisted of 70ml Sequagel (6%, 19:1

Acrylamide:Bisacrylamide, 1 x TBE), to which was added 560pl 10% APS. The gel was quickly poured using a 50ml syringe, with the gel plates lying horizontally. Two vinyl sharks tooth combs were placed beside each other with the flat surface approximately 0.5cm into the gel. Clips were placed across the top of the plates until the gel was set.

Once set, the gel was placed in a vertical BRL tanked and clamped into place. The combs were removed and the gel was pre-run in 1 x TBE at 65W for 1 hour. Following this the combs were inverted so that the teeth just penetrated the gel to form wells. The wells were washed out with 1 x TBE prior to loading.

PCR amplification was performed using direct incorporation of a-^^P- dCTP as described in section 2.2.2. 5pl of PCR product was diluted in

51^1 formamide dye, denatured for 3 minutes at 95°C, held on ice and

3.5|il loaded onto the gel. The gel was run at 65W for about 2-3 hours at depending on the size of the PCR product. Following this, the gel plates were prised apart and the gel blotted onto a piece of 3MM Whatman paper, covered with cling film and dried at 80°C for 1 hour. The dried gel was exposed to X-ray film, overnight at room temperature.

Microsatellite markers labelled with fluorescent dyes

PCR reaction was carried out in a 25)liI volume as described in section

2.2.2. Gel plates were washed in alconox, rinsed thoroughly in milliQ water and left to air-dry. Once dry the plates were then assembled in the cassette with 0.2mm spacers and the plates clamped. Polyacrylamide gel electrophoresis was carried out on the ABI 377. Gel mix consisted of 37ml diluent (Sequagel), 8ml concentrate (Sequagel), and 5ml of 10x

TBE. Prior to pouring, 400pl 10% APS and 20pl TEMED was added. The gel was quickly poured using a 50ml syringe, with the gel plates lying horizontally, and left to set for 1 hour prior to running. A 1 in 10 dilution of each PCR product was made, and 2pl of this diluted product was added to 2pl of size standard, GS350 (PE Applied Biosystems). These samples were denatured at 95°C for 3 minutes and held on ice prior to loading 2.5pl per lane. The gel was electrophoresed for 2.5 hours under the parameters stipulated by Genescan programme GS2400C. Following this, the data was collected and analysed using the Genescan version 2.0 and Genotyper software, supplied by the manufacturer (PE Applied Biosystems).

2 2.4.4. Sequencing products

Direct sequencing was performed on an ABI 377 automated DNA sequencer (PE Applied Biosystems). Gel plates were prepared as described for microsatellite markers labelled with fluorescent dyes, in section 2.2.4.3. Gel mix consisted of 18g ultrapure urea, 5.2ml acrylamide [19:1], 25ml milliQ water and 0.5g Amberlite in order to deionise the solution. Once dissolved, 5ml of 10 x TBE was added and the gel mix filtered and de-gassed using a 0.2pM cellulose nitrate filter. Prior to pouring, 250pl freshly-made 10% APS and 35pl TEMED was added. The gel was quickly poured using a 50ml syringe, with the gel plates lying horizontally. Once set, the gel was wrapped in cling film and allowed to stand for 2 hours prior to running.

The gel was electrophoresed for 7 hours using the following parameters: Dye set: DT dRset-Any; Matrix: dRHOD; Plate check: Plate check A; Pre­ run module: Seq PR 36A-1200 and Run module: Seq run 36E-1200. The following day, the sequence run was analysed using the Sequence Analysis version 3.0 and Sequence Navigator f software (PE Applied

Biosystems).

2.2.4.S. Single Stranded conformation (SSC) analysis