High Pure PCR Template Preparation Kit

Figure 1: PCR amplification of long templates from nucleic acid samples obtained with the High Pure PCR Template Preparation Kit. Nucleic acids were prepared from blood or cultured human K562 cells. 250 ng of each preparation was amplified using a tissue-plasminogen-activator (tPA)-specific primer and either the Expand Long Template PCR System or the Expand 20 kb^PLUS PCR System. Lanes 2. 6.3 kb; obtained from blood and amplified using Expand Long Template PCR System buffer 1 3. 15 kb; obtained from blood and amplified using Expand Long Template PCR System buffer 3 4. 23 kb; obtained from blood and amplified using Expand Long Template PCR System buffer 3 5. 28 kb; obtained from K562 cells and amplified using Expand 20 kb^PLUS PCR System Result: All High Pure preparations contained genomic DNA that yielded a distinct band of the expected size.

Figure 2: Southern blot analysis of nucleic acid samples obtained with the High Pure PCR Template Preparation Kit. Nucleic acids were prepared from cultured mammalian cells (10⁶ K562 cells) and from calf thymus tissue (25 mg). Aliquots of the preparations were digested with Eco RI, purified (see below), separated electrophoretically, and transferred to a membrane by Southern blotting. The blot was hybridized to a DIG-labeled α-actin antisense RNA probe according to a standard procedure. The hybridized bands (i.e., α-actin sequences) were detected with an alkaline phosphatase-labeled anti-DIG antibody and visualized via chemiluminescence with CSPD substrate. The blot was exposed to X-ray film for 1 hour. Lanes 1, 9. DNA Molecular Weight Marker III 2, 3. 1 μg and 3 μg samples of K562 genomic DNA, purified with the High Pure PCR Product Purification Kit after Eco RI digestion 4-6. 0.3 μg, 1 μg, and 3 μg samples of K562 genomic DNA, purified by conventional phenol/chloroform extraction and ethanol precipitation after Eco RI digestion 7, 8. 0.4 μg and 4 μg samples of calf thymus genomic DNA, purified by conventional phenol/chloroform extraction and ethanol precipitation after Eco RI digestion Result: All genomic DNA samples isolated using the High Pure PCR Template Preparation Kit were readily and completely cut by Eco RI, were suitable for Southern blot analysis, and contained the expected small and large fragments of the actin gene.

Figure 3: PCR amplification of nucleic acids prepared from paraffin-embedded tissue with the High Pure PCR Template Preparation Kit. Lanes 1. DNA Molecular Weight Marker VIII 2. Androgen receptor amplicon from a full tissue section 3. Androgen receptor amplicon from half of a full tissue section Result: The 263 bp androgen receptor amplicon was clearly visible in both samples.

Figure 4: Analysis of PCR products specific for S. aureus (left) and various lactobacilli (right). PCR templates were prepared from various samples (prepared as described below) with the High Pure PCR Template Preparation Kit, and analyzed by agarose gel electrophoresis. Left Panel Lanes 1,12. DNA Molecular Weight Marker, 1 kb ladder. 2. Negative control (no DNA). 3-5.S. aureus; lysis without lysostaphin; incubation periods of 15, 30, and 60 minutes 6-8. S. aureus; lysis with 10 μ/mL lysostaphin; incubation periods of 15, 30, and 60 minutes 9-11. S. aureus; lysis with 25 μ/mL lysostaphin; incubation periods of 15, 30, and 60 minutes Right Panel Lanes 13,18. DNA Molecular Weight Marker, phage lambda DNA, Hind III digest. 14. L. casei 15. L. curvatus 16. L. sakei 17. Negative control (no DNA)

Note: The lysis buffer contained 10 mg/mL lysozyme for lactobacilli and 1 mg/mL lysozyme for staphylococci together with the amounts of mutanolysin (MU) or lysostaphin (Ly) shown in the first column. The percentages refer to the DNA yield obtained with the highest mutanolysin or lysostaphin concentration and a 30-minute incubation period.

Typical DNA Recovery

Principle Flow Chart