Purification of DNA

2.2.1 Phenol chlorophorm extraction

For small volumes of DNA, the extraction volume was increased to 200 pi with Ix TE. Larger volumes were extracted directly. An equal volume o f phenol: chloroform: isoamyl alcohol (25:24:1) was mixed gently into the solution by inversion. The samples were than centrifuged at 6000 rpm for 3 minutes to separate the aqueous and organic layers. The top aqueous layer was transferred to a clean tube and the phenol extraction was repeated if necessary, depending on the protein content o f the sample. An equal volume o f chloroform was added, mixed and re-spun. The aqueous samples were transferred to a fresh tube for ethanol precipitation.

2.2.2 Ethanol precipitation

For standard DNA precipitations, two volumes o f absolute ethanol and 1/10^ volume o f 3 M sodium acetate were added to the solution and placed at -80°C for 30 minutes to allow the DNA to precipitate. The samples were than spun at 13,000 rpm for 15 minutes. The pellet o f DNA was washed with 200 pi 70% ethanol and the samples were centrifuged again

for 5 minutes. The pellet was either air-dried or was placed under vacuum for approximately 10 minutes. The samples were resuspended in distilled water.

2.2.3 Sephacryl -S200 and S400 HR columns (Pharmacia)

S200 HR columns were used for desalting, buffer exchange, and the removal of labelled or unlabelled nucleotides from DNA solutions, providing the DNA fragment was >100 bp. S400 HR columns serve the same purpose, but also remove primers or excess primer dimers from PCR products prior to sequencing. The Sephacryl HR resin (sephacryl equilibrated in TE buffer, pH 7.6) was resuspended in the column by gentle vortexing. The cap was loosened and the base snapped off before placing in an open topped eppendorf. This was spun at 3000 rpm for 1 minute to compact the gel matrix. The column was transferred to a fresh tube, and 20-25 pi of the DNA sample was added to the bed o f resin. The sample was collected at the bottom of the tube by centrifuging again for 1 min at 3000 rpm.

2.2.4 Centricon 100 spin columns (Centricon)

These columns were used for the purification o f PCR products prior to ABI sequencing. Columns were assembled according to manufacturer’s guidelines and 5-10 pi o f PCR product in 2 ml of sterile distilled water was added to the upper reservoir o f each column. These were centrifuged at 1000 g for 15 minutes (allowing the DNA to remain on the membrane while all unincorporated primers and dNTPs pass through). The column was than inverted and centrifuged for 5 minutes at 3000 g allowing the DNA elute to be collected. The purified products were directly used for cycle sequencing.

2.2.5 The enzymatic method {Exo\ and SAP)

This is an alternative quicker and cheaper method for purifying PCR products prior to sequencing. An aliquot of the amplification product (8 pi) was purified by the addition o f 1 U shrimp alkaline phosphatase (SAP; Amersham Life Science) and 1 U Exonuclease I (United States Biochemical) in SAP buffer. Samples were incubated for 30 minutes at 37°C followed by 15 minutes at 80°C. The purified products were directly used for cycle sequencing.

2.2.6 QIAquick spin columns (QIAGEN)

This method is designed to purify PCR products ranging from 100 bp to 10 kb eliminating primers, nucleotides, polymerases and salts.

Five volumes o f buffer PB (provided) were added to 1 volume o f the PCR reaction mix, the sample was than applied to a QIAquick spin column previously placed in a 2 ml collection tube, and centrifuged for 30-60 seconds at 13,000 rpm. The flow-through was discarded and the column was placed back into the same tube. To wash, 0.75 ml o f buffer PE (provided) was added to the column, followed by centrifugation for 30-60 seconds at 13,000 rpm. After discarding the flow-through the column was centrifuged for an additional minute at maximum speed to eliminate any residual ethanol from buffer PE. The column was than placed in a clean 1.5 tube. To elute DNA, 30 pi o f buffer EB (provided) was added to the centre o f the column, left to stand for 1 minute and spun for 1 minute at 13,000 rpm. The eluted DNA was directly used for cycle sequencing.

2.2.6.2 QIAquick gel extraction

This method is designed to extract and purify DNA of 70 bp to 10 kb from agarose gel in TAE buffer.

The DNA fragment o f interest was excised from the agarose gel with a clean, sharp scalpel, placed in a 1.5 tube and weighed. Three volumes of buffer QG (provided) were added to 1 volume o f gel and the mixture was than incubated at 50°C for 10 minutes or until the gel slice was completely dissolved. Occasionally, during the incubation time, the tube was vortexed to help dissolve the gel slice. After it was completely dissolved, 1 gel volume o f isopropanol was added to the sample and mixed. To bind DNA, the sample was applied to a QIAquick column previously placed in a 2 ml collection tube and spun for 1 minute at maximum speed. After discarding the flow-through and placing the column back in the same collection tube, 0.5 ml o f buffer QG was added to the column followed by 1 minute o f centrifugation at 13,000 rpm. The flow-through was again discarded. To wash, 0.75 ml o f buffer PE was added to the column, followed by centrifugation for 30-60 seconds at 13,000 rpm. After discarding the flow-through, the column was centrifuged for an additional minute at maximum speed to eliminate any residual ethanol from buffer PE. The column was than

placed in a clean 1.5 ml tube. To elute DNA, 30 pi o f buffer EB was added to the centre o f the column, left to stand for 1 minute and spun for 1 minute at 13,000 rpm. The eluted DNA was directly used for cycle sequencing.