Gotaq hot start kit

Manufactured by Promega

Sourced in United States

The GoTaq Hot Start kit is a reagent for performing polymerase chain reaction (PCR) amplification. It contains a modified DNA polymerase that is inactive at lower temperatures, preventing non-specific amplification during setup and initial heating steps.

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Lab products found in correlation

Mirneasy mini kit, by Qiagen (1 mentions)

Close spelling variants of this product

GoTaq G2 Hot Start Green Master Mix kit GoTaq G2 Hot Start PCR kit GoTaq hot start polymerase kit

4 protocols using gotaq hot start kit

RNA Extraction and Gene Expression Analysis

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Total RNA from cell lines and tumors were extracted using RNeasy or miRNeasy Mini kits (Qiagen), respectively. CDNA were prepared as described earlier [47 (link)]. PCR using GoTaq Hot Start Kit (Promega) were performed with following primers: Ngfr-for 5'-GAATGCGAGGAGATCCCTGG-3', Ngrf-rev 5'-GGAGCAATAGACAGGAATGAGG-3', Snai1-for 5'-CACCCATACAGGTGAGAAGC-3', Snai1-rev 5'-TGTCCTGGATGACAGAACCA-3', Nefh-for 5'-GCAGCCAAAGTGAACACAGA-3, Nefh-rev 5'-CTGAATAGCGTCCTGGTAGG-3', Mash1-for 5'-TTGAACTCTATGGCGGGTTC-3', Mash1-rev 5'-GCCATCCTGCTTCCAAAGTC-3', human ALK-for 5'-TGTTGCCTCTCCTCGATGTG-3', human ALK-rev TGTCTTCTCCGCTAATGGTG-3', murine Alk-for 5'-TGCCAGAAGTGTGTTCAGAAC-3', murine Alk-rev 5'-CCCTTCCATGAAGGCTTCAG-3'. Primers for Snai2, Sox9, Sox10, Gfap, Gapdh were described [29 (link)]. Cycling reactions were 2min at 95°C followed by 35 cycles of 30s at 95°C, 30s at 60 to 65°C and 30s at 72°C, followed by 5min at 72°C.

Montavon G., Jauquier N., Coulon A., Peuchmaur M., Flahaut M., Bourloud K.B., Yan P., Delattre O., Sommer L., Joseph J.M., Janoueix-Lerosey I., Gross N, & Mühlethaler-Mottet A. (2014). Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells. Oncotarget, 5(12), 4452-4466.

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Sanger Sequencing Validation of TMEM87B Mutation

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Validation of the mutation was done by PCR amplification followed by Sanger sequencing using genomic DNA from the patient and his parents (50 ng), and forward primer of 5′-TGGAAGACTTACTGGTTGGAAAG-3′ and reverse primer of 5′-CCCAGTTCAGTCATTCGCTATC-3′. The PCR conditions were as follows: Promega GoTaq Hot Start kit with 1× Master Mix and 400 nm of each primer. PCR began with an initial cycle at 95°C for 3 min, followed by 30 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min, finishing with extension at 72°C for 5 min. Amplified PCR products were sequenced using the PCR primers as sequencing primers on an ABI PRISM 3730xl at a commercial sequencing facility. Variations detected in TMEM87B were named using cDNA accession number NM_032824.2.

Yu H.C., Coughlin C.R., Geiger E.A., Salvador B.J., Elias E.R., Cavanaugh J.L., Chatfield K.C., Miyamoto S.D, & Shaikh T.H. (2016). Discovery of a potentially deleterious variant in TMEM87B in a patient with a hemizygous 2q13 microdeletion suggests a recessive condition characterized by congenital heart disease and restrictive cardiomyopathy. Cold Spring Harbor Molecular Case Studies, 2(3), a000844.

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Genetic Analysis of FOXL2 Mutations

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Genomic DNA samples from the patient and his parents (100 ng) were amplified by using two sets of primers specific to FOXL2 (NM_023067.3). Forward primer 1: 5′-GAGCTTAGGAAAGCGAAAAAGCAC AGAGGG-3′, reverse primer1: 5′-GAAGACATGTTCGAGAAGGGCAACTACCG-3′, forward primer 2: 5′-GTTGAGGAAGCCAGACTGCAGGTACTTGGG-3′, and reverse primer 2: 5′-TCTCCAGAAGTTTGAGACTTGGCCGTAAGC-3′. PCR reaction and conditions were as follows: Promega (Madison, WI) GoTaq Hot Start kit with 1× Master Mix and 400 nM of each primer. PCR began with an initial cycle at 95°C for 3 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 second and 72°C for 1 minute, finishing with extension at 72°C for 5 minutes. Amplified PCR products were sequenced using the PCR primers as sequencing primers on an ABI (Carlsbad, CA) PRISM 3730xl at a commercial sequencing facility. High resolution copy number analysis was performed using Affymetrix (Santa Clara, CA) SNP 500K arrays as previously described [Haldeman-Englert et al., 2010 (link)].

Yu H.C., Geiger E.A., Medne L., Zackai E.H, & Shaikh T.H. (2014). An Individual with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) and Additional Features Expands the Phenotype Associated with Mutations in KAT6B. American journal of medical genetics. Part A, 0(4), 950-957.

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Validation of Genetic Variants in CARS2 Gene

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Variants identified in CARS2 (NM_024537.2, ENST00000 257347) were further validated by Sanger sequencing in the subject and his parents. Primers were designed to amplify and sequence exons 6 and 7 of CARS2 where the identified sequence variants are located in the proband. Primers were designed to amplify and sequence all exon and intron boundaries of CARS2 in the additional 15 subjects. Genomic DNA (100 ng) was amplified using PCR, with reactions and conditions as follows: Promega GoTaq Hot Start' kit (Promega, Madison, Wisconsin, USA) with 1× Master Mix and 400 nM of each primer. The PCR began with an initial cycle at 95°C for 3 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min, finishing with extension at 72°C for 5 min. Amplified PCR products were sequenced using the PCR primers as sequencing primers on an ABI (Life Technologies, Carsbad, California, USA) PRISM 3730xl at a commercial Clinical Laboratory Improvement Amendments (CLIA)-certified sequencing facility.

Coughlin CR 2.n.d., Scharer G.H., Friederich M.W., Yu H.C., Geiger E.A., Creadon-Swindell G., Collins A.E., Vanlander A.V., Coster R.V., Powell C.A., Swanson M.A., Minczuk M., Van Hove J.L, & Shaikh T.H. (2015). Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder. Journal of medical genetics, 52(8).

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