Mytaq extract pcr kit

Villin:Cre mice were obtained from Jackson Laboratories and Zfp36^fl/fl mice have been described previously^{19 (link)}. Intestinal epithelial cell-specific TTP knockout mice, termed ΔIEC, were generated by crossing Zfp36^fl/fl mice with Villin:Cre mice expressing CRE recombinase under the control of the Villin promoter. All experiments were performed using Zfp36^fl/fl littermates as controls. Mice were routinely genotyped by isolating genomic DNA from a 2 mm tail biopsy as described previously⁵⁸ . The floxed Zfp36 allele was amplified as described previously and the Cre transgene was amplified as recommended by Jackson Laboratories using the indicated primers (Table S2)^{19 (link)}. Tissue-specific knockout was confirmed by first using the MyTaq Extract-PCR Kit (Bioline) to isolate genomic DNA from 2 cm of colon tissue. Subsequently, multiplexed PCR was used to amplify either the floxed or deleted Zfp36 alleles with the oligonucleotides described previously and listed in Table S2^{19 (link)}. Unless otherwise indicated, all experiments were performed on 6-8-week-old mice. All animal breeding and experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Pennsylvania State University College of Medicine. All experiments were performed in accordance with relevant guidelines and regulations provided by the IACUC.

First, genomic DNA was isolated from 1 × 10⁶ cells using the MyTaq Extract-PCR Kit (Bioline GmbH, Luckenwalde, Germany) or innuPREP DNA Mini Kit (Analytik Jena, Jena, Germany) following the manufacturer’s recommendations. Next, a 403 bp DNA fragment within the lsr gene that contains the LSR-specific gRNA binding site was amplified by PCR with oligonucleotides 5′-GTC​CAA​CCC​CTA​CCA​CGT​GGT​G-3′ and 5′- GCT​TTC​AGA​TGG​GGA​CTC​CAG​G-3′ and by using genomic DNA as template (Hemmasi et al., 2015 (link)). Eventually, the PCR product was purified with the my-Budget Double Pure Kit (BioBudget Technologies GmbH, Krefeld, Germany) and subjected to DNA sequencing (Eurofins Genomics Europe Sequencing GmbH, Konstanz, Germany). Indels in the sequenced DNA fragments were identified by performing sequence alignments with the lsr reference sequence.

A male chimeric Gsdme KO mouse, Gsdme^{tm1a(KOMP)Wtsi}, was designed in 2011 by KOMP (Knockout Mouse Project; University of California, Davis, CA 95616, USA). This mouse was crossed with a female WT C57BL/6N mouse (Charles River Laboratories [LA2230391], Wilmington, MA, USA) to obtain a male heterozygous Gsdme KO mouse. This mouse was crossed with female WT C57BL/6N mice to obtain more heterozygous Gsdme KO mice. In turn, these heterozygous Gsdme KO mice were crossed to obtain Gsdme KO and WT mice.
When mice were four weeks old, they were weaned and ear punched. DNA was extracted from the ear punches (MyTaq™ Extract-PCR Kit, Bioline, Memphis, TN, USA) and mice were genotyped by PCR. The genotyping protocol for both Gsdme and Apc^1638N can be found in the Supplementary Material (Tables S6–S11).
Mice were screened twice a week for abnormal behavior, weight loss, diarrhea, rectal bleeding and prolapse. If mice had lost ≥ 20% of their body weight, they were sacrificed.

The genotyping was performed as described^{51 (link)}. The DNA was extracted from ear biopsies by using MyTaq™ Extract-PCR Kit (Bioline, Luckenwalde, Germany) following the manufacturer’s instructions. The genomic DNA was subjected to PCR amplification to identify the mutations.
The presence of the CaSR flox allele was tested by performing a PCR amplification (2 min at 94 °C; 30 sec at 94 °C followed by 30 sec at 60 °C for 34 cycles and 1 min at 68 °C) using Casr-F and Casr-E3-R primers (see the table below). Zygosity was tested by digestion of the PCR product with the SalI enzyme (NEB Biolabs) for 3 h at 37 °C, which was then run on a 1% agarose gel. Homozygous mice generated 2 bands (400 bp and 250 bp), and heterozygous mice generated 3 bands (650 bp, 400 bp and 250 bp). The Cre transgene was detected by PCR using Cre FW and Cre RV primers (see the table below).

Primer name	Primer sequence (3′→5′)
CaSR-F	5′-GACTTGCTATGTAGCCCAGAACTG-3′
CaSR-E3-R	5′-AAGGGATGTGCTCGGAGCA-3′
Cre Fw	5′-AAATTGCCAGGATCAGGGTTAAAG-3′
Cre Rv	5′-AGAGTCATCCTTAGCGCCGTAAAT-3′