We targeted chr4.hg19:46918163-47005187 encompassing 74kb of GABRA4 (NM_000809) and 10kb upstream and 5kb downstream of the gene with long-range PCR. Ten tiled primer pairs each amplifying ~8.7kb were designed using Primer3 software (http://frodo.wi.mit.edu ) [32 (link), 33 (link)] (Online Resource 1) and synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA). PCR was performed using the Expand Long Range dNTPack PCR kit (Roche Applied Science, Indianapolis, IN, USA) on Veriti 96-Well Fast Thermal Cyclers (Life Technologies, Carlsbad, CA, USA). PCR products were visualized on 0.75% agarose-ethidium bromide gel and primer dimer and nonspecific products removed with the NucleoFast 96 PCR kit (Machery-Nagel, Bethlehem, PA, USA) following standard manufacturer protocol for a vacuum manifold.
Expand long range dntpack pcr kit
Manufactured by Roche
Sourced in United States
The Expand Long Range dNTPack PCR kit is a reagent kit designed for performing long-range PCR amplification. The kit contains a proprietary enzyme mix and a set of dNTPs optimized for efficient amplification of long DNA fragments.
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Lab products found in correlation
3 protocols using expand long range dntpack pcr kit
1
Targeted GABRA4 Gene Sequencing
2
Amplification and Sequencing of Centromeric K111
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Centromeric K111 s were amplified by PCR with the primers P1, which binds to K111 flanking centromeric repeat CER:D22Z3, and primer P4, which binds to the K111 gag gene to produce a ~ 1.6 Kb amplification product [25 (link)]. The PCR was carried out using the Expand Long Range dNTPack PCR kit (Roche Applied Science, Indianapolis, IN). PCR was performed at a final volume of 50 μl using an initial step of 92 °C for 2 min, followed by 35 PCR cycles consisting of denaturation at 92 °C for 15 s, annealing at 55 °C for 30 s, and extension at 68 °C for 5 min. Amplification products were confirmed by sequencing [25 (link)].
3
Amplification and Sequencing of K111 Insertions
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K111 insertions were amplified by PCR using the Expand Long Range dNTPack PCR kit (Roche Applied Science, Indianapolis, IN) as described27 (link). K111 5′ and 3′ LTRs and accompanying flanking regions were amplified. PCR was performed using an initial step of 94 °C for 2 min followed by 35 cycles consisting of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 68 °C for 5 min. The amplification products were cloned into the topo TA vector (Invitrogen, Carlsbad, CA) and sequenced.
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