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==================================
*Arabidopsis thaliana* cell lines
==================================

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

* :doc:`/cell_lines/rpc00008`

* :doc:`/cell_lines/rpc00050`

* :doc:`/cell_lines/rpc00055`

* :doc:`/cell_lines/rpc00056`

* :doc:`/cell_lines/rpc00103`

* :doc:`/cell_lines/rpc00104`

Genotypes
=========

  .. csv-table::
     :header: "Cell line", "rbcL", "*GNOM*", "*TOM*", "Transgene insertion", "InDel/SSR"
     :widths: auto

     "\ :doc:`T87 </genotyping/g_rpc00008>`\ ", "*Arabidopsis thaliana*", "---", "---", "---", "T87"
     "\ :doc:`YG1 </genotyping/g_rpc00050>`\ ", "*Arabidopsis thaliana*", "---", "---", "T-DNA (not detected)", "At tom/YG1"
     "\ :doc:`gnom </genotyping/g_rpc00055>`\ ", "*Arabidopsis thaliana*", "*gnom*", "---", "---", "---"
     "\ :doc:`At tom </genotyping/g_rpc00056>`\ ", "*Arabidopsis thaliana*", "---", "*tom1-2* |br| *tom3-1*", "T-DNA (homozygous)", "---"
     "\ :doc:`MM2d </genotyping/g_rpc00103>`\ ", "*Arabidopsis thaliana*", "---", "---", "---", "MM2d"
     "\ :doc:`MM2d-LS </genotyping/g_rpc00104>`\ ", "*Arabidopsis thaliana*", "---", "---", "---", "MM2d-LS"

Protocols
=========

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

  :doc:`_rbcL`

DNA sequencing --- *GNOM* gene
------------------------------

#. PCR

   * Primer sets 《:doc:`_primers`》

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

          "*GNOM*", "gnom_At1g133980_F", "gnom_At1g133980_R", "500 bp"

   * 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 genome 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.

   * *gnom* | *GNOM* At1g133980: p.Arg647Ter (c.1939C>T)

.. 要確認： PCR reaction mixture、PCR reaction

DNA sequencing --- *TOM* genes
------------------------------

#. PCR

   * Primer sets 《:doc:`_primers`》

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

          "*TOM1*", "tom1-2_F", "tom1-2_R", "450 bp"
          "*TOM3*", "Tom3-1_F", "Tom3-1_R", "550 bp"

   * 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 genome 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.

   * *tom1-2* | *TOM1* At4g21790: p.Trp68Ter (c.204G>A)

   * *tom3-1* | *TOM3* At2g02180: p.Trp45Ter (c.135G>A)

.. 要確認： PCR reaction mixture、PCR reaction

Transgene insertion --- T-DNA (GABI-Kat 046D04)
-----------------------------------------------

#. PCR

   * Primer sets 《:doc:`_primers`》

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

          "Left border", "2625", "046D04_flankingseq_side", "900 bp"
          "Zygosity", "046D04_RBside", "046D04_flankingseq_side", "Not detected (850 bp)"

   * 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 genome 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 size of the PCR product.

.. 要確認： PCR reaction mixture、PCR reaction

Fragment analysis --- InDel (1 loci)
------------------------------------

#. PCR

   * Primer sets 《:doc:`_primers`》

     * 3483/3484 primer mix

         .. csv-table::
            :header: "Primer name", "Concentration"
            :widths: auto

            "3483", "1 µM"
            "3484", "0.1 µM"

     * uni-FAM primer: Fluorescent labeled primer at 5′ end with 6-FAM™

         .. csv-table::
            :header: "Primer name", "Concentration"
            :widths: auto

            "uni-FAM", "10 µM"

   * 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 the PCR product with the Applied Biosystems™ 3130xl Genetic Analyzer

#. Data analysis with the GeneMapper® Software

Fragment analysis --- SSR (16 loci)
-----------------------------------

#. PCR with each of the primer mix

   * Primer sets: Fluorescent labeled primers at 5′ end

       +----------+-------+-----------------+-------------+
       |Primer mix|Marker |Fluorescent label|Concentration|
       +==========+=======+=================+=============+
       |PS1       |nga280 |6-FAM™           |1 µM         |
       +          +-------+-----------------+-------------+
       |          |nga1126|VIC\ :sup:`®`    |2.5 µM       |
       +          +-------+-----------------+-------------+
       |          |nga6   |NED™             |1 µM         |
       +          +-------+-----------------+-------------+
       |          |nga1111|PET™             |1 µM         |
       +----------+-------+-----------------+-------------+
       |PS2       |nga106 |6-FAM™           |1 µM         |
       +          +-------+-----------------+-------------+
       |          |nga63  |VIC\ :sup:`®`    |2.5 µM       |
       +          +-------+-----------------+-------------+
       |          |nga168 |NED™             |1 µM         |
       +          +-------+-----------------+-------------+
       |          |nga172 |PET™             |1 µM         |
       +----------+-------+-----------------+-------------+
       |PS3       |nga1107|6-FAM™           |1 µM         |
       +          +-------+-----------------+-------------+
       |          |nga129 |VIC\ :sup:`®`    |0.5 µM       |
       +          +-------+-----------------+-------------+
       |          |nga162 |NED™             |0.5 µM       |
       +          +-------+-----------------+-------------+
       |          |nga1139|PET™             |1 µM         |
       +----------+-------+-----------------+-------------+
       |PS4       |nga111 |6-FAM™           |2 µM         |
       +          +-------+-----------------+-------------+
       |          |nga1145|VIC\ :sup:`®`    |1 µM         |
       +          +-------+-----------------+-------------+
       |          |nga158 |NED™             |1 µM         |
       +          +-------+-----------------+-------------+
       |          |Det1.2 |PET™             |1 µM         |
       +----------+-------+-----------------+-------------+

   * PCR reaction mixture

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

          "2×QIAGEN Multiplex", "10 µL"
          "Primer mix", "2 µL"
          "Template DNA", "1 µL"
          "Sterile distilled water", "7 µL"

   * PCR reaction

       +--------------+------+---------+
       |Temperature   |Time  |Cycles   |
       +==============+======+=========+
       |95°C          |15 min|         |
       +--------------+------+---------+
       |94°C          |30 sec|35 cycles|
       +--------------+------+         +
       |54--62°C [#]_ |90 sec|         |
       +--------------+------+         +
       |72°C          |1 min |         |
       +--------------+------+---------+
       |60°C          |30 min|         |
       +--------------+------+---------+

       .. [#] * PS1: 54°C

              * PS2: 60°C/55°C

              * PS3: 58°C

              * PS4: 62°C

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

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