For 5’-RACE analysis, we used one mouse from CRISPR-Cas9-treated and control groups. The total RNA was isolated from frozen muscle samples and reverse transcribed by Mint revertase (Evrogen, SK001) with the specific primer to Dmd cDNA, Dp71-fl-Rev. Step-out amplification of upstream Dmd sequences was performed with the Mint RACE primer set (Evrogen, SK004) and reverse primers designed for exons 64–66 (Dp71-5′ rev1-3; Table S3 ). The obtained bulk PCR products were purified from gel and ligated into the pAL-TA vector (Evrogen, TA002) with further transformation of ligation mixes into XL1-Blue cells (Evrogen, CC001). Ten clones for each group were sent for Sanger sequencing.
Pal ta vector
Manufactured by Evrogen
The PAL-TA vector is a plasmid designed for the expression of target proteins in bacterial systems. It provides a platform for the cloning and expression of desired genes, allowing for the production of recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mint revertase, by Evrogen (1 mentions)
Mint race primer set, by Evrogen (1 mentions)
Xl1 blue cells, by Evrogen (1 mentions)
Vector nti advance 10, by Thermo Fisher Scientific (1 mentions)
Plasmid miniprep kit, by Evrogen (1 mentions)
Cleanup mini kit, by Evrogen (1 mentions)
Mint cdna synthesis kit, by Evrogen (1 mentions)
3 protocols using pal ta vector
1
RNA analysis of Duchenne muscular dystrophy
2
Isolation and Characterization of Radish CLE Genes
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Total DNA of R. sativus was isolated from radish seedlings using the cetyltrimethylammonium bromide (CTAB) method [42 (link)]. To amplify the fragments of radish CLE genes, PCR was performed with primers designed for the conserve regions of corresponding genes of Arabidopsis thaliana and Brassica rapa using a VectorNTI Advance_10 (Invitrogen, United States) program (Additional file 4 : Table S2). PCR-products were separated by electrophoresis in 1 % agarose gel containing ethidium bromide (0.1 %). Target fragments were isolated from the gel using a Cleanup Mini Kit (Evrogen, Russia) according to the manufacturer’s instructions and cloned into the pAL-TA vector (Evrogen, Russia). Transformation of chemically competent cells of Escherichia coli strain DH5α was carried outaccording to the protocol described in [43 (link)]. Transformants were selected on solidified LB medium containing 100 mg/L ampicillin and X-Gal. Plasmid DNA of selected transformant clones was isolated by Plasmid Miniprep Kit (Evrogen, Russia) and sequenced in SPbU Research Park, Center of Molecular and Cell Technologies. Sequence aligning of Arabidopsis and radish genes was performed using the AlignX program of VectorNTI Advance_10 (Invitrogen) software.
3
Generating cDNA Expression Library from Xenopus Embryos
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cDNA expression library was prepared by the SMART approach from total embryonic RNA with a Mint cDNA synthesis kit (Evrogen, Russia). cDNA gene fragments were isolated from the library by PCR with gene-specific primers (see Table 1 ). Primers were designed based upon sequences obtained from the sequenced transcriptome (Illumina) of D. pumila35 (link). Amplified fragments were cloned into the pAL-TA vector (Evrogen, Russia). Digoxygenine‐labeled antisense RNA probes were generated from gene fragments, which were amplified from plasmids with D. pumila genes.
PCR and qPCR primers used in this study.
| Gene | Direct primer 5' ‑> 3' | Reverse primer 5' ‑> 3' |
|---|---|---|
| DpBra1 in situ probe | TTGGTGGCGACAGCGAAGAA | CGGCCACGTGTTGTTTTGAATG |
| DpBra2 in situ probe | GAACGGAGAGGGCAAAGACAAAC | GACGGCGAATATGGGGAACAAAT |
| DpBra3 in situ probe | AATAATTCTTCACCGTCCAACAGG | CGCGCTTTTCGTGATAGATAGG |
XlTubb2b.S (β-tubulin) qPCR | GATCCTACCGGCAGTTACCA | TGACAGAGTCCATTGTGCCT |
| XlActc1.L (cardiac actin) qPCR | CTATGTGGCTTTGGACTTTGAG | GCTGTTGTAGGTAGTTTCATG GA |
| XlMyod1.S qPCR | AGTGACAGCCCAAATGACTC | AGAAGGGATGGTGATTACTCTC |
| XlEF1a qPCR | CCCTGCTGGAAGCTCTTGAC | GGACACCAGTCTCCACACGA |
| XlODC qPCR | GGGCTGGATCGTATCGTAGA | TGCCAGTGTGGTCTTGACAT |
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