Probe templates in the range of 300–400 bp were PCR amplified from P. axillaris cDNA for PaxH4 (Peaxi162Scf00679g00110), NAM (Peaxi162Scf00069g01832), and PaxGATA19 (Peaxi162Scf00366g00123), using the v1.6.2 reference genome to design primers in Primer3 (Table S3 ; Fig. S3 ). Each template was ligated with pGEM-T (Promega, Madison, WI, USA), cloned into DH5alpha cells, and sequence confirmed after colony PCR using M13F and M13R primers. Depending on the orientation of the insert, T7 or SP6 RNA polymerase (Sigma-Aldrich, St. Louis, USA) was used with digoxigenin RNA labeling mix (Sigma-Aldrich, St. Louis, MO, USA) to generate sense and antisense probes. Hybridization of each probe within serial P. axillaris floral tissue sections followed Preston and Kellogg (2007) (link) with a few modifications. Specifically, probes were not hydrolyzed, protein degradation was carried out for 30 min at 37°C with 25 μg/ml proteinase K (Sigma-Aldrich, St. Louis, MO, USA), and probe hybridization was done overnight at 52–55°C.
T7 or sp6 rna polymerase
Manufactured by Merck Group
Sourced in United States
The T7 or SP6 RNA polymerase is an enzyme used in molecular biology laboratories for the in vitro synthesis of RNA. It is derived from the bacteriophage T7 or SP6 and recognizes specific promoter sequences to initiate transcription, allowing for the production of RNA from DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Merck Group (1 mentions)
Pgem t, by Promega (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Alkaline phosphatase conjugated anti dig antibody, by Roche (1 mentions)
Nitroblue tetrazolium 5 bromo 4 chloro 3 indolyl phosphate, by Roche (1 mentions)
Pcrii vector, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Promega (1 mentions)
2 protocols using t7 or sp6 rna polymerase
1
In Situ Hybridization of Petunia Floral Genes
2
In Situ Hybridization Probing of Mouse Sf3b4
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A specific primer set (5 0 -CGGTGCTGCTGCTTAGAGAC-3 0 and 5 0 -AGGATGAGGCATTCCAGGAG-3 0 ) was used to yield a 1070 bp length of the mouse Sf3b4 gene that completely includes the RNA probe region used in the previous study. 28 These fragments subcloned into pGEM-T easy (Promega) or pCRII vector (Invitrogen). Digoxigenin (DIG)-labeled antisense or sense RNA probes were generated with T7 or SP6 RNA polymerase (Sigma-Aldrich). E8.5, 9.5, and 10.5 mouse embryos were fixed with 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS) overnight (O/N) on ice. These samples were dehydrated by methanol and stored at -20 C until use for whole-mount in situ hybridization, or embedded in O.C.T. compound (Sakura Finetek, Japan) and stored at -80 C until use for section in situ hybridization. The frozen samples sliced into 8 μm frontal sections were hybridized with DIG-labeled probes at 70 C. After stringent washing and incubating with alkaline phosphatase-conjugated anti-DIG antibody (1:5000 [vol/vol] dilution, Roche Diagnostics, Germany), signals were detected using nitro blue tetrazolium/5-bromo-4chloro-3-indolyl-phosphate (Roche Diagnostics).
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