All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Stanford University. The following mice were purchased from The Jackson Laboratory (Sacramento, CA): FVB (Stock Number # 001800), Gli1tm3(cre/ERT2)Alj/J(Gli1-CreERT2 (Stock number # 007913)) and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J(Stock number # 007909). To obtain Gli1CreERT2;tdTomato (denoted as Gli1-CE;tdT) mice, heterozygous Gli1-CreERT2 mice were crossed with homozygous B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J mice. To confirm mouse genotypes, tail samples of pups under 21 days of age were analyzed by PCR. Tail samples were digested using Quick Extract DNA Extraction Solution (Fisher, cat # QE09050) and PCR was performed on DNA samples using a 2700 ABI thermocycler (Applied Biosystems). The program of the thermocycler was as follows: 95 – 5 minutes, 35 cycles of :95 – 45 seconds, 55 – 45 seconds, 72 – 45 seconds, then 72 – 10 minutes and 10 – hold. List of Primers are shown in Table 1 .
Abi 2700 thermocycler
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
The ABI 2700 thermocycler is a laboratory instrument designed for performing polymerase chain reaction (PCR) amplification of DNA samples. The core function of the ABI 2700 is to precisely control the temperature cycling required for the various steps of the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nucleospin rna mini kit, by Macherey-Nagel (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Abi prism 3100, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Abi 3730xl dna analyzer, by Sangon (1 mentions)
Ex taq dna polymerase kit, by Takara Bio (1 mentions)
Hi di formamide, by Thermo Fisher Scientific (1 mentions)
3130 genetic analyser, by Thermo Fisher Scientific (1 mentions)
5 protocols using abi 2700 thermocycler
1
Generating Transgenic Gli1-CE;tdT Mice
2
RNA Extraction and cDNA Synthesis
3
Molecular Detection of ESBL and Quinolone Resistance
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Whole-cell DNAs were extracted using QIAamp DNA Mini Kit (Qiagen, Les Ulis, France). The blaCTX-M, blaSHV, blaTEM, minor ESBL and quinolone resistant qnrA, B and S genes were searched for by PCR and subsequently characterized by Sanger sequencing. PCR experiments were performed on an ABI 2700 thermocycler (Applied Biosystems, Les Ulis, France) using laboratory-designed primers [19 (link)]. Both strands of the PCR products, were sequenced using laboratory-designed primers with an automated sequencer (ABI PRISM 3100; Applied Biosystems). The nucleotide and the deduced protein sequences were analyzed using software available at the National Center of Biotechnology Information website (http://www.ncbi.nlm.nih.gov ).
4
Mitogenome Sequencing of Anemonefishes
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Total genomic DNA was extracted from the muscle by standard phenol-chloroform procedures [24 ]. The mitogenomes of anemonefishes were determined using eight consensus primer pairs with a long PCR technique (Table 2 ) [25 (link)]. PCR amplifications were carried out on an ABI 2700 Thermo Cycler (www.appliedbiosystems.com ) in 25 μl reaction volumes, by using Takara Ex Taq DNA polymerase kit (www.takara.com ) as indicated by the manufacturer. PCR amplifications were under the following standard cycle: one denaturation step at 94°C for 3 min, 30 cycles of 94°C for 45 s, 50~60°C for 1 min and 72°C for 1 min 30 s, followed by a final elongation step at 72°C for 10 min. PCR products were sequenced on an ABI3730XL DNA Analyzer (Sangon Biotech, www.sangon.com )
5
CALR Mutation Detection Protocol
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Published primers [2] (link) targeting exon 9 of CALR were used (Applied Biosystems, UK). A primer-based sizing assay was used to detect CALR mutations, the forward primer (GGCAAGGCCCTGAGGTGT) being labelled with 6-carboxyflourescein (6-FAM). The reverse primer sequence was GGCCTCAGTCCAGCCCTG.
Hot-start PCR was used. The amplicons labelled with 6-FAM were electrophoresed in a 3130 Genetic Analyser (Applied Biosystems, UK), as well as on agarose gel.
A mixture of 10 µL Hi-Di Formamide and 0.5 µL GeneScan LIZ 500 (size standard), both from Life Technologies, USA, was prepared for capillary electrophoresis, of which 10 µL was added RESEARCH to 1.5 µL PCR product. Nested PCR was performed for the samples subjected to gel electrophoresis, whereby the PCR products were amplified for a second time in an ABI 2700 thermocycler (Applied Biosystems, USA). PCR products were separated on a 2.5% agarose gel and analysed under ultraviolet light. Samples positive for a mutation were band-stabbed and placed in TE -1 buffer (EDTA 0.5M, Tris 1M) overnight at 4°C. Four µL of the supernatant was used for sequencing.
The wild-type allele has 267 base pairs. [8] Any fragments that differed in size to the wild-type allele carried a mutation.
Hot-start PCR was used. The amplicons labelled with 6-FAM were electrophoresed in a 3130 Genetic Analyser (Applied Biosystems, UK), as well as on agarose gel.
A mixture of 10 µL Hi-Di Formamide and 0.5 µL GeneScan LIZ 500 (size standard), both from Life Technologies, USA, was prepared for capillary electrophoresis, of which 10 µL was added RESEARCH to 1.5 µL PCR product. Nested PCR was performed for the samples subjected to gel electrophoresis, whereby the PCR products were amplified for a second time in an ABI 2700 thermocycler (Applied Biosystems, USA). PCR products were separated on a 2.5% agarose gel and analysed under ultraviolet light. Samples positive for a mutation were band-stabbed and placed in TE -1 buffer (EDTA 0.5M, Tris 1M) overnight at 4°C. Four µL of the supernatant was used for sequencing.
The wild-type allele has 267 base pairs. [8] Any fragments that differed in size to the wild-type allele carried a mutation.
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