====================
Transgene insertion
====================

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

* :doc:`/cell_lines/rpc00014`

* :doc:`/cell_lines/rpc00015`

* :doc:`/cell_lines/rpc00039`

* :doc:`/cell_lines/rpc00040`

* :doc:`/cell_lines/rpc00041`

* :doc:`/cell_lines/rpc00042`

* :doc:`/cell_lines/rpc00062`

* :doc:`/cell_lines/rpc00069`

* :doc:`/cell_lines/rpc00091`

* :doc:`/cell_lines/rpc00093`

* :doc:`/cell_lines/rpc00095`

Genotypes
==========

  .. csv-table::
     :header: "Cell line", "Transgene insertion"
     :widths: auto

     "\ :doc:`CRA <../../genotyping/g_rpc00014>`\ ", "Not detected (*tmr*)"
     "\ :doc:`V208 <../../genotyping/g_rpc00015>`\ ", "*tmr*"
     "\ :doc:`GV7 <../../genotyping/g_rpc00039>`\ ", "35Spro-GFP-AtVam3p"
     "\ :doc:`GF11 <../../genotyping/g_rpc00040>`\ ", "35Spro-GFP-AtFim1"
     "\ :doc:`GT16 <../../genotyping/g_rpc00041>`\ ", "35Spro-GFP-tubulin"
     "\ :doc:`TBY2-AtRER1B <../../genotyping/g_rpc00042>`\ ", "35Spro-GFP-AtRer1B"
     "\ :doc:`BY-TIPG <../../genotyping/g_rpc00062>`\ ", "35Spro-NtTIP1;1-GFP"
     "\ :doc:`156-S <../../genotyping/g_rpc00069>`\ ", "35Spro-CjSMT"
     "\ :doc:`TBY2-31/ST <../../genotyping/g_rpc00091>`\ ", "35Spro-GFP-AtSYP31/35Spro-ST-mRFP"
     "\ :doc:`TBY2-41/ST <../../genotyping/g_rpc00093>`\ ", "35Spro-GFP-AtSYP41/35Spro-ST-mRFP"
     "\ :doc:`TBY2-R31 <../../genotyping/g_rpc00095>`\ ", "35Spro-mRFP-AtSYP31"

Protocol
========

#. PCR

   * Primer sets 《:doc:`_primers`》

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

           "CRA", "*tmr*", "35Spro_F3", "GV7_R", "Not detected (370 bp)"
           "V208", "*tmr*", "35Spro_F3", "GV7_R", "370 bp"
           "GV7", "35Spro-GFP-AtVam3p", "35Spro_F3", "GV7_R", "1.5 kbp"
           "GV7", "35Spro-GFP-AtVam3p", "GFP_F", "GV7_R", "1.3 kbp"
           "GF11", "35Spro-GFP-AtFim1", "35Spro_F3", "GF11_R", "2.0 kbp"
           "GF11", "35Spro-GFP-AtFim1", "GFP_F", "GF11_R", "1.7 kbp"
           "GT16", "35Spro-GFP-tubulin", "35Spro_F3", "GT16_R", "2.1 kbp"
           "GT16", "35Spro-GFP-tubulin", "GFP_F", "GT16_R", "1.9 kbp"
           "TBY2-AtRER1B", "35Spro-GFP-AtRer1B", "35Spro_F3", "RER1B_R", "1.4 kbp"
           "TBY2-AtRER1B", "35Spro-GFP-AtRer1B", "GFP_494_F", "RER1B_R", "800 bp"
           "TBY2-AtRER1B", "35Spro-GFP-AtRer1B", "GFP_F", "RER1B_R", "1.2 kbp"
           "TBY2-AtRER1B", "35Spro-GFP-AtRer1B", "GFPS65T_F", "RER1B_R1", "565 bp"
           "BY-TIPG", "35Spro-NtTIP1;1-GFP", "35Spro_F3", "GFP_R", "1.3 kbp"
           "BY-TIPG", "35Spro-NtTIP1;1-GFP", "NtTIP1_F", "GFP_R", "1.0 kbp"
           "156-S", "35Spro-CjSMT", "35Spro_F2", "SMT_R", "1.5 kbp"
           "156-S", "35Spro-CjSMT", "35Spro_F2", "NOSterR1", "2.0 kbp"
           "TBY2-31/ST", "35Spro-GFP-AtSYP31", "35Spro_F5", "SYP31stop_R", "1.8 kbp"
           "TBY2-31/ST", "35Spro-ST-mRFP", "35Spro_F2", "mRFPstop_R", "1.1 kbp"
           "TBY2-41/ST", "35Spro-GFP-AtSYP41", "35Spro_F5", "SYP41stop_R", "1.8 kbp"
           "TBY2-41/ST", "35Spro-ST-mRFP", "35Spro_F2", "mRFPstop_R", "1.1 kbp"
           "TBY2-R31", "35Spro-mRFP-AtSYP31", "35Spro_F5", "SYP31stop_R", "1.8 kbp"

   * PCR reaction mixture

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

           "TaKaRa Ex Taq™ Premix", "5 µL"
           "Forward primer (10 µM)", "0.5 µL"
           "Reverse primer (10 µM)", "0.5 µL"
           "Template DNA", "1 µL"
           "Sterile distilled water", "3 µL"

   * PCR reaction

       +-----------+-----------+---------+
       |Temperature|Time       |Cycles   |
       +===========+===========+=========+
       |94°C       |1 min      |         |
       +-----------+-----------+---------+
       |94°C       |30 sec     |35 cycles|
       +-----------+-----------+         +
       |55°C       |30 sec     |         |
       +-----------+-----------+         +
       |72°C       |1 min/1 kbp|         |
       +-----------+-----------+---------+
       |72°C       |10 min     |         |
       +-----------+-----------+---------+

#. Check the size of the PCR product.

#. If necessary, check the DNA sequence of the PCR product.