Exosap it pcr product cleanup

The studied SNPs were TLR4 rs4986790 on chromosome 9q33.1, TLR9 rs187084 on chromosome 3p21.2 and TLR2 rs4696480 on chromosome 4q31.3. Genotyping was performed in genomic DNA extracted from peripheral blood samples by using the QIAamp DNA Blood Mini Kit (Qiagen) according to manufacturer’s recommendations. Partial amplification of the genes containing the different polymorphisms was performed using specific primers as follows: TLR4 rs4986790 forward 5′-CTACCAAGCCTTGAGTTTCTAG-3′, reverse 5′-AAGCTCAGATCTAAATACCT-3′; TLR9 rs187084 forward 5′-CATTCATTCAGCCTTCACTC-3′, reverse: 5′-ATGTGCTGTTCCCTCTGC-3′; TLR2 rs4696480 forward 5′-GGGACAAGAATAAAGTACATAGTTG-3′, reverse 5′-GGCTGTACCCTCATAAATGGA-3′. PCR product sizes (110 bp, 419 bp and 297 bp, respectively) were purified using ExoSAP-IT PCR Product Cleanup (Affymetrix). The incidence of polymorphisms was detected by nucleotide sequencing of PCR products using the same primers as for the amplification. The sequencing process was performed by Secugen SL. The results were analysed with FinchTV software version 1.5 (Geospiza). The incidence of all three polymorphisms in control population was obtained from The 1000 Genomes Project Consortium29 (link) (www.1000genomes.org).

Samples were recruited from the formalin-fixed, paraffin embedded surgical specimens of OCCCs. DNA was extracted using the Gentra Puregene Tissue Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. Polymerase chain-reaction (PCR) was performed using the following specific primers designed for exon 12: 5′-GCTCCCTATATCCCAATGGA-3′ (forward) and 5′-CAGTGGTGGCAGTGGTAGTG-3′ (reverse). The PCR conditions applied were: 1 cycle of 95 °C for 2 min, followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 1 min at 72 °C with final extension at 72 °C for 10 min. For each assay, a negative control (without DNA template) was added to monitor PCR contaminations. After confirming the integrity of the amplicons, all PCR products were further purified using ExoSAP-IT PCR Product Clean-up (Affymetrix, Santa Clara, CA, USA) for commercial sequencing. The sequencing primer was the same as the forward primer used for the PCR reaction. Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used for reading sequences on the chromatograms.

We designed PCR primers to amplify candidate variants identified by exome sequencing as well as to screen all coding regions of PJA1 (NM_145119) and MSX2 (NM_002449). Genomic DNA from peripheral blood was amplified by PCR. Genomic DNA samples, OKI‐005‐5 and OKI‐005‐6, were extracted from saliva using Oragene DISCOVER (DNA Genotek Inc). Primer sequences and PCR conditions are available upon request. The PCR products were purified using ExoSAP‐IT PCR product Cleanup (Affymetrix) and analyzed by direct sequencing using an ABI PRISM 3730xl Genetic Analyzer.

In order to study Tngfp insertion sites LMPCR was used according to the protocol described by Prod’hom et al. (1998) (link). This technique allowed the amplification of both ends of the transposon. Briefly, the DNA was digested with the SalI enzyme, the fragments of DNA were ligated with an adapter Sal, containing Salgd and Salpt sequences (Table 2A), and the resulting template was then digested with SalI. The PCR was performed using IS2 and gfp1 primers (specific for Tngfp and directed outward) and the common linker primer Salgd (Table 2A). DNA was denatured by incubating the mixture at 95°C for 5 min. Amplification was achieved using 35 cycles of PCR (95°C for 30 s, specific annealing temperature for 30 s and 72°C for 90 s), followed by a final extension at 72°C for 10 m. Amplified products were separated by standard horizontal gel electrophoresis in a 1.5% agarose gel in TBE buffer (90 mM Tris, 90 mM boric acid, and 2 mM EDTA) and were stained using ethidium bromide. PCR products were purified using the GFX^TM PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech Inc.) followed by ExoSAP-IT^® PCR product clean up (Affymetrix).
The amplified products were sequenced with the corresponding oligonucleotides using CNIO³ service and further checked for homology at Tuberculist⁴, Bovilist⁵ and NCBI⁶ database BLAST analysis.

DNA eluates were subjected to long-template PCR to amplify the complete HPV16 genome. Two overlapping fragments encompassing the complete genome were generated using primer combinations F1832/R6382 and F6201/R2915 (reference 24 (link) and data not shown). PCR was performed using TaKaRa PrimeStar GXL according to the manufacturer's protocol. The cycling conditions consisted of initial incubation at 98°C for 8 min followed by 38 cycles of 98°C denaturation for 15 s, 55°C annealing for 30 s, and 68°C elongation for 5 min and a final elongation step at 68°C for 15 min.
PCR product amplification was verified on the Lonza FlashGel system. If both fragments amplified successfully, samples were treated with ExoSap-It PCR product cleanup (Affymetrix) according to the manufacturer's protocol. If amplification failed for the initial sample, the follow-up sample was excluded from further analyses. If amplification succeeded for the initial sample but failed for the follow-up sample, the infections were sequenced without follow-up. Purified PCR products were subjected to Sanger sequencing using 45 unique primers for HPV16, covering the complete genome in both forward and reverse directions (references 24 (link)– (link)28 (link) and data not shown).

Sanger sequencing was used to resequence the complete exons 26 and 28 and confirm all putative mutations discovered with Ion Torrent sequencing. The human genomic sequence was obtained from genomic build GRCh37p13. Primers for the 52 different exons of the
VWFgene and exon 7 of the
ABOgene (
Supplementary Table S2) were designed using NCBI Primer-BLAST (
http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi) and ordered from DNA Technology A/S (Risskov, Denmark). The samples were sequenced using Big Dye Terminator chemistry, v3.1 (Applied Biosystems, Foster City, California, United States) on an ABI 3130xl sequencer. PCR was performed using KAPA Taq HotStart DNA PCR kit (KAPA Biosystems, Cape Town, South Africa). The primary PCR products were purified with ExoSAP-IT PCR Product Cleanup (Affymetrix, Santa Clara, California, United States), unincorporated dye terminators removed by BigDye XTerminator Purification Kit and sequence data assembled and compared using SeqScape (Applied Biosystems).