All experiments were approved by the local animal care and use committee in accordance with the German animal protection law. The generation of functional Ambra1 null mutant mice via inactivation of the Ambra1 gene has been described in detail elsewhere (Fimia et al., 2007 (link)). Ambra1+/− and wildtype (WT, Ambra1+/+) littermates of both genders with a >99% C57BL/6N genetic background were used for behavioral and biochemical studies. They were obtained from male Ambra1+/− × female WT C57BL/6N breeding pairs. Genotypes of the offspring were analyzed by PCR of tail genomic DNA using the following primers: Ambra1 forward primer 5′-GAA AAG CTC CCC ATC TTT TCT T-3′, Ambra1 reverse primer 5′-ATC CCA AGG GCA GTA GAG TTC-3′ (yielding a 3 kb product in Ambra1+/−), interleukin-2 (IL-2) forward primer 5′-CTA GGC CAC AGA ATT GAA AGA TCT-3′, and IL-2 reverse primer 5′-GTA GGT GGA AAT TCT AGC ATC ATC C-3′ (yielding 350 bp product in all samples for an internal control). PCR amplification of the DNA was carried out with Failsafe enzyme with PreMix D (Epicentre, Madison, WI, USA) under the following conditions: 3 min, 94°C (1 cycle); 30 s, 94°C; 45 s, 57°C; 2 min 30 s, 72°C (40 cycles), followed by final extension at 72°C for 5 min. Final PCR products were run on a 1.5% agarose gel and stained with ethidium bromide.
Premix d
Manufactured by Illumina
Sourced in United States
Premix D is a reagent mixture used in genetic analysis workflows. It is designed to facilitate specific and efficient amplification of target DNA sequences. The core function of Premix D is to provide the necessary components for performing polymerase chain reaction (PCR) or other amplification-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Aligner v9, by CodonCode (9 mentions)
Taq polymerase, by Merck Group (3 mentions)
Exosap it express, by Thermo Fisher Scientific (2 mentions)
Simpliamp thermocycler, by Thermo Fisher Scientific (2 mentions)
Platinum taq, by Thermo Fisher Scientific (1 mentions)
Magna pure compact, by Roche (1 mentions)
Exosap, by Thermo Fisher Scientific (1 mentions)
Mutation surveyor software, by SoftGenetics (1 mentions)
5 protocols using premix d
1
Genetic Characterization of Ambra1 Mice
2
Familial Variant Sanger Sequencing
3
Sequencing Protocol for INSR Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from leukocytes in blood using MagNAPure Compact instrument (Roche Applied Science, Indianapolis, IN). Nucleic acid sequencing for the INSR gene coding region was performed by standard dideoxy termination. PCR primers were developed for the 22 exons of the INSR isoform containing exon 11 (NM_002082 ). Eighteen sets of PCR primers used in this validation were previously published [20] (link), however, in the current study four sets of primers were re-designed to optimize PCR and sequencing results (see Table 1 ). We added another internal primer set to exon three interior to the homopolymer region to obtain cleaner sequence. In addition, exons 18 and 19 were consolidated into one amplicon. polymerase chain reaction of the 22 coding exons of the INSR gene was performed using M13-tailed primers Premix D (Epicentre, Madison, WI), and Platinum Taq (Invitrogen, Carlsbad, CA) using PCR conditions shown in Table 2 below. Unused PCR primers and unincorporated nucleotides were inactivated by incubation with ExoSAP (USB Corporation, Cleveland, OH). Bidirectional DNA sequencing was performed with BigDye Terminator chemistry (ABI, Foster City, CA) and M13 primers (IDT, Coralville, IA) and the product was analyzed on the ABI 3730. Data analysis was performed using Mutation Surveyor software (SoftGenetics, State College, PA) and GenBank reference sequence NG_008852.1 .
4
Genetic Variant Screening for AIS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Variants appearing in multiple families that were present in the GO functional categories for “cytoskeleton” or “extracellular matrix” (or related terms) were prioritized for genotyping. Additional enrolled affected and unaffected family members were sequenced at the variant site by the Sanger method to establish whether the variant segregated with the AIS phenotype.
PCR was conducted in 20 µL reactions containing 10 µL Premix D (Epicentre Biotechnologies, Madison, WI, USA), 0.2 µL Taq Polymerase (Sigma, St. Louis, MO, USA), 60 ng genomic DNA, and 10 µM Forward and Reverse Primers. PCR reactions were run on a SimpliAmp Thermocycler (Fisher Scientific, Waltham, MA, USA) with a touchdown PCR protocol [38 (link)]. Primer sequences were obtained from Integrated DNA Technologies and are provided inSupplementary Files S2 . Sanger sequencing was performed by Quintara Biosciences and chromatograms were analyzed using the CodonCode Aligner v9.0 (CodonCode Corporation, Centerville, MA, USA, https://www.codoncode.com/index.htm (accessed on 16 June 2021)).
Pedigrees for each sequenced family are provided inSupplementary Files S1 and were created using PedigreeXP software (PC Pal, https://www.pedigreexp.com (accessed on 16 June 2021)).
PCR was conducted in 20 µL reactions containing 10 µL Premix D (Epicentre Biotechnologies, Madison, WI, USA), 0.2 µL Taq Polymerase (Sigma, St. Louis, MO, USA), 60 ng genomic DNA, and 10 µM Forward and Reverse Primers. PCR reactions were run on a SimpliAmp Thermocycler (Fisher Scientific, Waltham, MA, USA) with a touchdown PCR protocol [38 (link)]. Primer sequences were obtained from Integrated DNA Technologies and are provided in
Pedigrees for each sequenced family are provided in
5
Family-based Sanger Sequencing of Exome Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA from additional affected and unaffected family members was Sanger sequenced across the variants of interest detected through exome sequencing. Primers were designed using Primer3 and obtained from Integrated DNA Technologies. PCR was conducted in 20 µL reactions with 10 µL Premix D (Epicentre Biotechnologies), 0.2 µL Taq Polymerase (Sigma), 20 ng genomic DNA, and 20 µM Forward and Reverse Primers. PCR reactions were run with a touchdown PCR protocol on a SimpliAmp Thermocycler (Fisher Scienti c). Annealing temperatures were set with the delta function to begin at 65°C and decrease by 0.3 °C each cycle, ending at 55 °C. PCR reactions were cleaned up for Sanger sequencing using ExoSAP-IT Express (Thermo Fisher). Sanger sequencing was performed by Quintara Biosciences and chromatograms were analyzed using the CodonCode Aligner v9.0 (CodonCode Corporation, https://www.codoncode.com/index.htm).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!