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*Nicotiana* (tobacco) cell lines
=================================

Plant cell lines
================

* :doc:`/cell_lines/rpc00001`

* :doc:`/cell_lines/rpc00009`

* :doc:`/cell_lines/rpc00035`

* :doc:`/cell_lines/rpc00036`

* :doc:`/cell_lines/rpc00039`

* :doc:`/cell_lines/rpc00040`

* :doc:`/cell_lines/rpc00041`

* :doc:`/cell_lines/rpc00042`

* :doc:`/cell_lines/rpc00043`

* :doc:`/cell_lines/rpc00044`

* :doc:`/cell_lines/rpc00062`

* :doc:`/cell_lines/rpc00084`

* :doc:`/cell_lines/rpc00085`

* :doc:`/cell_lines/rpc00086`

* :doc:`/cell_lines/rpc00091`

* :doc:`/cell_lines/rpc00093`

* :doc:`/cell_lines/rpc00095`

Genotypes
=========

  .. csv-table::
     :header: "Cell line", "rbcL", "Transgene insertion", "*psbA*", "SSR"
     :widths: auto

     "\ :doc:`BY-2 </genotyping/g_rpc00001>`\ ", "*Nicotiana tabacum*", "---", "---", "BY-2"
     "\ :doc:`T-13 </genotyping/g_rpc00009>`\ ", "*Nicotiana tabacum*", "---", "---", "T-13"
     "\ :doc:`Xan-1 </genotyping/g_rpc00035>`\ ", "*Nicotiana tabacum*", "---", "---", "Xan-1"
     "\ :doc:`BY-2H </genotyping/g_rpc00036>`\ ", "*Nicotiana tabacum*", "---", "---", "BY-2H"
     "\ :doc:`GV7 </genotyping/g_rpc00039>`\ ", "*Nicotiana tabacum*", "35Spro-GFP-AtVam3p", "---", "---"
     "\ :doc:`GF11 </genotyping/g_rpc00040>`\ ", "*Nicotiana tabacum*", "35Spro-GFP-AtFim1", "---", "---"
     "\ :doc:`GT16 </genotyping/g_rpc00041>`\ ", "*Nicotiana tabacum*", "35Spro-GFP-tubulin", "---", "---"
     "\ :doc:`TBY2-AtRER1B </genotyping/g_rpc00042>`\ ", "*Nicotiana tabacum*", "35Spro-GFP-AtRer1B", "---", "---"
     "\ :doc:`3n-3 </genotyping/g_rpc00043>`\ ", "*Nicotiana tabacum*", "---", "---", "3n-3"
     "\ :doc:`G89 </genotyping/g_rpc00044>`\ ", "*Nicotiana glauca*", "---", "---", "G89"
     "\ :doc:`BY-TIPG </genotyping/g_rpc00062>`\ ", "*Nicotiana tabacum*", "35Spro-NtTIP1;1-GFP", "---", "---"
     "\ :doc:`NI </genotyping/g_rpc00084>`\ ", "*Nicotiana tabacum*", "---", "---", "NI"
     "\ :doc:`ATR-r </genotyping/g_rpc00085>`\ ", "*Nicotiana tabacum*", "---", "p.Ser264Thr", "ATR-r"
     "\ :doc:`NaCl-r </genotyping/g_rpc00086>`\ ", "*Nicotiana tabacum*", "---", "---", "NaCl-r"
     "\ :doc:`TBY2-31/ST </genotyping/g_rpc00091>`\ ", "*Nicotiana tabacum*", "35Spro-GFP-AtSYP31 |br| 35Spro-ST-mRFP", "---", "---"
     "\ :doc:`TBY2-41/ST </genotyping/g_rpc00093>`\ ", "*Nicotiana tabacum*", "35Spro-GFP-AtSYP41 |br| 35Spro-ST-mRFP", "---", "---"
     "\ :doc:`TBY2-R31 </genotyping/g_rpc00095>`\ ", "*Nicotiana tabacum*", "35Spro-mRFP-AtSYP31", "---", "---"

Protocols
=========

DNA barcoding --- rbcL
----------------------

  :doc:`_rbcL`

Transgene insertion
-------------------

  :doc:`_transgene`

DNA sequencing --- *psbA* gene
------------------------------

#. PCR

   * Primer set 《:doc:`_primers`》

       .. csv-table::
          :header: "Target", "Forward primer", "Reverse primer", "Product size"
          :widths: auto

          "*psbA*", "PCRpsbALEFT", "PCRpsbARIGHT", ""

   * PCR reaction mixture

       .. csv-table::
          :header: "Component", "10 µL reaction"
          :widths: auto

          "TaKaRa Ex Taq™ Premix", "5 µL"
          "Forward primer (100 µM)", "0.125 µL"
          "Reverse primer (100 µM)", "0.125 µL"
          "Template DNA", "1 µL"
          "Sterile distilled water", "3.75 µL"

   * PCR reaction

       +-----------+------+---------+
       |Temperature|Time  |Cycles   |
       +===========+======+=========+
       |94°C       |1 min |         |
       +-----------+------+---------+
       |94°C       |30 sec|35 cycles|
       +-----------+------+         +
       |55°C       |30 sec|         |
       +-----------+------+         +
       |72°C       |1 min |         |
       +-----------+------+---------+
       |72°C       |7 min |         |
       +-----------+------+---------+

#. Check the DNA sequence of the PCR product.

   * *psbA*: p.Ser264Thr (c.791G>C)

.. 要確認： PCR reaction mixture、PCR reaction

Fragment analysis --- SSR (3 loci)
----------------------------------

#. PCR with each of the primer mix

   * Primer sets 《:doc:`_primers`》

       +----------+------------+-------------+
       |Primer mix|Primer  name|Concentration|
       +==========+============+=============+
       |3493/3494 |3493        |1 µM         |
       +          +------------+-------------+
       |          |3494        |0.1 µM       |
       +----------+------------+-------------+
       |3495/3496 |3495        |1 µM         |
       +          +------------+-------------+
       |          |3496        |0.1 µM       |
       +----------+------------+-------------+
       |3497/3498 |3497        |1 µM         |
       +          +------------+-------------+
       |          |3498        |0.1 µM       |
       +----------+------------+-------------+
       |          |uni-FAM [#]_|10 µM        |
       +----------+------------+-------------+

       .. [#] Fluorescent labeled primer at 5′ end with 6-FAM™

   * PCR reaction mixture

       .. csv-table::
          :header: "Component", "10 µL reaction"
          :widths: auto

          "AmpliTaq Gold™ 360 Master Mix", "5 µL"
          "uni-FAM", "0.2 µL"
          "Primer mix", "2 µL"
          "Template DNA", "1 µL"
          "Sterile distilled water", "1.8 µL"

   * PCR reaction

       +-----------+------+---------+
       |Temperature|Time  |Cycles   |
       +===========+======+=========+
       |95°C       |10 min|         |
       +-----------+------+---------+
       |95°C       |30 sec|40 cycles|
       +-----------+------+         +
       |55°C       |30 sec|         |
       +-----------+------+         +
       |72°C       |30 sec|         |
       +-----------+------+---------+
       |72°C       |10 min|         |
       +-----------+------+---------+

#. Capillary electrophoresis of each of the PCR products with the Applied Biosystems™ 3130xl Genetic Analyzer

#. Data analysis with the GeneMapper\ :sup:`®` Software