===========================================
*Solanum lycopersicum* (tomato) cell lines
===========================================

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

* :doc:`/cell_lines/rpc00012`

* :doc:`/cell_lines/rpc00045`

* :doc:`/cell_lines/rpc00046`

Genotypes
=========

  .. csv-table::
     :header: "Cell line", "rbcL", "matK", "*Tm-1*"
     :widths: auto

     "\ :doc:`Sly-1 </genotyping/g_rpc00012>`\ ", "*Solanum lycopersicum*", "Sly-1", "---"
     "\ :doc:`GCR26 </genotyping/g_rpc00045>`\ ", "*Solanum lycopersicum*", "GCR", "+/+"
     "\ :doc:`GCR237 </genotyping/g_rpc00046>`\ ", "*Solanum lycopersicum*", "GCR", "*Tm-1*/*Tm-1*"

Protocols
=========

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

  :doc:`_rbcL`

DNA barcoding --- matK
----------------------

#. PCR

   * Primer set 《:doc:`_primers`》

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

          "matK", "matK_F", "matK_R", "900 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 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       |10 min|         |
       +-----------+------+---------+

#. Check the sequence of the PCR product.

   * NCBI BLAST search

   * Compare the DNA sequence with that of previous years.

DNA sequencing --- *Tm-1* gene
------------------------------

#. PCR

   * Primer set 《:doc:`_primers`》

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

          "*Tm-1*", "GCR_F2", "GCRseq_R1", "600 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 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.

.. 要確認： DNA sequencing --- PCR reaction mixture、PCR reaction