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Commit aefb5548 authored by Gesa Helmsorig's avatar Gesa Helmsorig
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Update file cloning_trafo_protocol.md

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**cloning**
## Transformation of barley plants
**Cloning of transformation vectors:**
- cloning of transformation vectors was performed according to the protocol by Kumar et al. (2018)
- sgRNAs were cloned into recipient vector pMGE599
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| 1 | 7-26, 97-116 | GGCGGTGGCGCAGCCGCGGATGG, GAGATCTATACCTACGAGGCCGG | pMGE599/7-97
| 2 | 1182-1201, 1222-1241 | GGTCAGCTACCCAGCCTGACTGG, TAATAAACTGCAGATTCTCA GGG | pMGE599/182-1222
**transformation**
**Transformation:**
- gp-fast plants were cultivated in long-day conditions (16h day / 8 night) in control temperature (20°C / 16°C)
- 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:**
**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. Plant Biotechnology Journal, 16(11): 1892–1903.
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