Alw26I (BsmAI) (10 U/µL)

Protocol tips

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	One μl of the products from the second reaction was digested at 37°C for 1 hour with 1 unit of enzyme (Alw26I and HpyAV for the codons 198 and 200) in a total volume of 15 μl. The products were subjected to electrophoresis in a 6.0% polyacrylamide gel (w/v) stained with GelRed (Biotium, USA).

Protocol tips
One μl of the products from the second reaction was digested at 37°C for 1 hour with 1 unit of enzyme (Alw26I and HpyAV for the codons 198 and 200) in a total volume of 15 μl. The products were subjected to electrophoresis in a 6.0% polyacrylamide gel (w/v) stained with GelRed (Biotium, USA).

Publication protocol

"In silico analysis of A. lumbricoides and N. americanus beta-tubulin nucleotide sequences retrieved from GenBank and WormBase ParaSite databases were performed using the NEBcutter V2.0 tool (http://www.labtools.us/nebcutter-v2-0/) in order to search for restriction sites that could be used to distinguish between the mutated and the wild type alleles (of 198 and 200 for N. americanus; and 167 and 198 for A. lumbricoides) so that a PCR-RFLP approach could be employed. The enzymes RsaI (Promega, USA) and BmsI (Thermo Fisher Scientific, USA) were chosen to differentiate between mutated and unmutated codons 167 and 198 of A. lumbricoides. Sites for enzymes Alw26I and HpyAV (Thermo Fisher Scientific, USA) were present in the N. americanus sequence and therefore used to distinguish between mutated and unmutated alleles for the codons 198 and 200 of N. americanus, respectively. A suitable enzyme that could differentiate between mutated and unmutated codon 200 of A. lumbricoides was not found. This same situation was observed for codon 167 of N. americanus.

For N. americanus, an initial PCR was performed using one primer pair to amplify the codons 198 and 200 (Fa198/200Na + Rab198/200Na; 325 bp) using GoTaq Green Master Mix (Promega, USA), with 0.2 μM of each primer and 4.2 μl of the buffer containing the DNA of a single egg. The cycling conditions followed the same parameters as described for the wild-type control synthesis. A semi-nested PCR was performed using 1 μl from the first PCR reaction employing primers Fb198/200Na + Rab198/200Na (315 pb), using the same PCR conditions as described above. One μl of the products from the second reaction was digested at 37°C for 1 hour with 1 unit of enzyme (Alw26I and HpyAV for the codons 198 and 200) in a total volume of 15 μl. The products were subjected to electrophoresis in a 6.0% polyacrylamide gel (w/v) stained with GelRed (Biotium, USA)."

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Alw26I (BsmAI) (10 U/µL) below.

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