Amplification and Sequencing of K111 Insertions

K111 insertions were amplified by PCR using the Expand Long Range dNTPack PCR kit (Roche Applied Science, Indianapolis, IN) as described^{27 (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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Saha A.K., Mourad M., Kaplan M.H., Chefetz I., Malek S.N., Buckanovich R., Markovitz D.M, & Contreras-Galindo R. (2019). The Genomic Landscape of Centromeres in Cancers. Scientific Reports, 9, 11259.

Publication 2019

Expand Long Range dNTPack PCR kit topo TA vector

Insertions Ltrs Topo Vector

Corresponding Organization :

Other organizations : University of Michigan–Ann Arbor, Hormel (United States), University of Minnesota, Magee-Womens Research Institute, University of Pittsburgh

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Variable analysis

independent variables

PCR amplification conditions (initial step of 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 68 °C for 5 min)

dependent variables

Amplification products of K111 5′ and 3′ LTRs and accompanying flanking regions

control variables

Use of the Expand Long Range dNTPack PCR kit (Roche Applied Science, Indianapolis, IN) for PCR amplification

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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