diff --git a/assays/DNA_extraction/isa.assay.xlsx b/assays/DNA_extraction/isa.assay.xlsx index fa568f021179b9a34cf6a441a7c4560bcb8e6bae..6a4e6d819baefd2c48387bac8e40a8acc99c14ab 100644 Binary files a/assays/DNA_extraction/isa.assay.xlsx and b/assays/DNA_extraction/isa.assay.xlsx differ diff --git a/assays/biparental_mapping/isa.assay.xlsx b/assays/biparental_mapping/isa.assay.xlsx index 4deb074ae9102a1aa31b405dbbf04a09a9ee32b6..86cf18bbaec63ee37127adc445443140a0afe41f 100644 Binary files a/assays/biparental_mapping/isa.assay.xlsx and b/assays/biparental_mapping/isa.assay.xlsx differ diff --git a/assays/cDNA_synthesis/isa.assay.xlsx b/assays/cDNA_synthesis/isa.assay.xlsx index 8afc2dea77b297e674df9fa278c154a8fba3d948..897d0e9596ad7ef56e9bedf03221b47b057de06c 100644 Binary files a/assays/cDNA_synthesis/isa.assay.xlsx and b/assays/cDNA_synthesis/isa.assay.xlsx differ diff --git a/assays/embryo_transformation/README.md b/assays/embryo_transformation/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/embryo_transformation/dataset/.gitkeep b/assays/embryo_transformation/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/embryo_transformation/dataset/transformation_T0_results.xlsx b/assays/embryo_transformation/dataset/transformation_T0_results.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..25c90dae811898a392421295cdb69f0d78f2f2f3 Binary files /dev/null and b/assays/embryo_transformation/dataset/transformation_T0_results.xlsx differ diff --git a/assays/embryo_transformation/isa.assay.xlsx b/assays/embryo_transformation/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..435f145f4d32fed2b0d3d3ba6d97bd824fd9a525 Binary files /dev/null and b/assays/embryo_transformation/isa.assay.xlsx differ diff --git a/assays/embryo_transformation/protocols/.gitkeep b/assays/embryo_transformation/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/embryo_transformation/protocols/QIAGEN_DNeasy-Plant-Mini.pdf b/assays/embryo_transformation/protocols/QIAGEN_DNeasy-Plant-Mini.pdf new file mode 100644 index 0000000000000000000000000000000000000000..0ddcd9b9decc71bd18483805dc49765ed843134a Binary files /dev/null and b/assays/embryo_transformation/protocols/QIAGEN_DNeasy-Plant-Mini.pdf differ diff --git a/assays/embryo_transformation/protocols/cloning_trafo_protocol.md b/assays/embryo_transformation/protocols/cloning_trafo_protocol.md new file mode 100644 index 0000000000000000000000000000000000000000..6e4c85e4c080edbe1acecfb0540909cf980ef95d --- /dev/null +++ b/assays/embryo_transformation/protocols/cloning_trafo_protocol.md @@ -0,0 +1,23 @@ +**cloning** + +- cloning of transformation vectors was performed according to the protocol by Kumar et al. (2018) +- sgRNAs were cloned into recipient vector pMGE599 +- two sgRNA combinations: + +| approach | covered CDS sequence (from start codon) | sgRNA sequence (incl. PAM, 5'-3') | construct name | +| - | - | - | - | +| 1 | 7-26, 97-116 | GGCGGTGGCGCAGCCGCGGATGG, GAGATCTATACCTACGAGGCCGG | pMGE599/7-97 +| 2 | 1182-1201, 1222-1241 | GGTCAGCTACCCAGCCTGACTGG, TAATAAACTGCAGATTCTCA GGG | pMGE599/182-1222 + +**transformation** + +- gp-fast plants were cultivated in long-day conditions (16h day / 8 night) in control temperature (20°C / 16°C) +- immature embryos were used for transformation according to Hensel et al. (2009) +- DNA was extracted from regenerated plants using QIAGEN_DNeasy-Plant-Mini protocol +- successful insertion of the transformation vector into the genome was tested by PCR using the primers Hyg-156 (5'-ACGCACAATCCCACTATCC-3') and Hyg-047 (5'-GTGTCGTCCATCACAGTTTG-3') + + +**References** +Hensel G, Kastner C, Oleszczuk S, Riechen J, Kumlehn J (2009) Agrobacterium-mediated gene transfer to cereal crop plants: Current protocols for barley, wheat, triticale, and maize. International Journal of Plant Genomics, 2009: 835608. + +Kumar N, Galli M, Ordon J, Stuttmann J, Kogel K-H, Imani J (2018) Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene-editing system. 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