Qiaquick pcr purification kit

SeqZip ligation reactions were amplified via PCR (Agilent, Herculase II Fusion DNA Polymerase, Catalog Number—600675) for 12 cycles using common primers. Reactions were resolved on a 5% polyacrylamide native gel, and DNA in the size range appropriate for full-length ligation products quantified by fluorescence imaging, cut and eluted from the gel, and precipitated. Equal DNA quantities based on the gel imaging were amplified for another 22 cycles using primers containing Illumina priming sequences with integrated barcodes. PCR products were purified (28104; Qiagen, QIAquick PCR Purification Kit) and quantified using a Bioanalyzer 2100 (Agilent) and High-Sensitivity DNA chip. Samples were mixed and submitted for sequencing on the MiSeq instrument using the paired-end 250 nt read option. Sequencing data are available at Short Read Archive accession SRP043516.

The amplified libraries were pooled in equimolar ratios, totaling a final amount of 2 µg. An established protocol was followed (Maricic, Whitten & Pääbo, 2010 (link)) except that synthesized oligonucleotide baits were used instead of PCR products and the EB volume for final elution using Qiagen MinElute column was 20 µL instead of 15 µL. After 2 days of hybridization and subsequent elution steps, 1 µL of the final eluate was quantified by qPCR and 5 µL (in total 15 µL) was amplified in triplicate using the same kit and cycling conditions as described in the NGS library preparation for second round amplification of Illumina indexed libraries. The pooled PCR products were purified using the QIAquick PCR Purification Kit and was measured using the Tapestation 2200 (Agilent Technologies Catalog G2964AA). Hybridization capture libraries were sequenced at the National High-throughput DNA Sequencing Centre, Copenhagen, Denmark using Illumina MiSeq Reagent Kit v2 (300 cycles).

To verify the RFLP patterns observed following restriction digestion, we used a nested PCR to amplify the full-length tpr genes separately for sequencing analysis[14 (link)]. Two μL of DNA was amplified in a 50 μL reaction with 2.5 units of TaKaRa polymerase (TaKaRa Bio, Inc., Mountain View, CA, USA), 1x PCR buffer, 200 nM dNTPs, and 0.2 μM of primers E1 and E2 for tpr E, G9.89 and G10.3026 for tpr G, or J2 and J3 for tpr J. Reaction conditions were as follows: 94°C for 1 min, then 35 cycles of 94°C for 30 sec, 60°C for 1 min, and 68°C for 3 min 30 sec, followed by a final extension at 68°C for 10 min. Two μL of outer PCR product were used for the nested reaction, which was carried out using the same conditions but with primers E3 and E4 for tpr E, G6.232 and TPRG8 for tpr G, and TPRJ9Fl and TPRJ11 for tpr J. Samples were run on an Agilent Bioanalyzer and subsequently purified using the QIAquick PCR Purification Kit for amplicon sequencing.
To verify the number of 60-bp repeats as well as the sequence of each repeat within arp, we purified all arp amplicons using the QIAquick PCR Purification Kit.

The PCR products (φX174) were amplified with a forward primer anchored to a T7 promoter sequence and a reverse primer anchored to an oligo(dT)₃₀ sequence. The sequences of the PCR primers and the sizes of the products are listed in Supplementary Table 6.
After examining the products with a bioanalyzer (Agilent), the excess primers were removed from the products using the QIAquick PCR Purification Kit. After ethanol precipitation, the DNA concentrations were measured by UV absorption.
RNA was synthesized by incubating 500 ng of PCR product at 37 °C for 1 hour in a 10-μl reaction mixture containing 90 nmol of dATP, dCTP, dGTP, and dUTP; 10 nmol of DTT; 1 μl of AmpliScribe T7-Flash Enzyme (EPICENTRE Biotechnologies); and 1 × AmpliScribe buffer. The RNA samples were purified by DNase and protease K treatments, followed by two extractions with phenol/chloroform according to the standard procedure. The residual dNTP in the purified RNA samples was then removed with an Oligotex-dT30 kit (TaKaRa Bio), and phenol/chloroform extraction was performed again. After ethanol precipitation, the pellets were resuspended in 100 μl of RT-PCR grade water (Life Technologies), and the RNA concentrations were measured by UV absorption. The subsequent procedures were identical to the ones previously described except that the qPCR primers described in Supplementary Table 7 were used.