PCR amplicons from each piglet from the small INDEL assay and the long range assay were TOPO cloned using the TOPO TA and TOPO XL kit, respectively (Thermo Scientific) by following standard protocol. Clones were propagated on Luria-Bertani (LB) agarose plates containing 50 μg/ml kanamycin and resistant recombinants were selected. Plasmids containing the TMPRSS2 amplicon were identified by either EcoRI or EagI digestion, and subsequent DNA agarose gel electrophoresis. Plasmids that contained a TMPRSS2 amplicon were DNA sequenced by the University of Missouri DNA core by using the TMPRSS2 F1 or TMPRSS2 3968F oligonucleotide, respectively. Sequences were aligned to the wild type (WT) TMPRSS2 gene in ApE (http://biologylabs.utah.edu/jorgensen/wayned/ape/ ) and INDELS were examined.
Topo xl kit
Manufactured by Thermo Fisher Scientific
The TOPO XL kit is a cloning system designed for the high-efficiency, directional cloning of PCR products. The kit includes pre-linearized, topoisomerase I-activated vector, and optimized reaction buffer for efficient ligation of PCR products into the vector.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using topo xl kit
1
TMPRSS2 Gene Variant Identification
2
TMPRSS2 Amplicon Sequencing Protocol
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PCR amplicons from each piglet from the small INDEL assay and the long range assay were TOPO cloned using the TOPO TA and TOPO XL kit, respectively (Thermo Scientific) by following standard protocol. Clones were propagated on Luria–Bertani (LB) agarose plates containing 50 μg/ml kanamycin and resistant recombinants were selected. Plasmids containing the TMPRSS2 amplicon were identified by either EcoRI or EagI digestion, and subsequent DNA agarose gel electrophoresis. Plasmids that contained a TMPRSS2 amplicon were DNA sequenced by the University of Missouri DNA core by using the TMPRSS2 F1 or TMPRSS2 3968F oligonucleotide, respectively. Sequences were aligned to the wild type (WT) TMPRSS2 gene in ApE (http://biologylabs.utah.edu/jorgensen/wayned/ape/ ) and INDELS were examined.
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