This is an advanced protocol. 

This is a transgenic cloning strategy for Arabidopsis thaliana to create a line for mutant rescue.  The development of this protocol is attributed to Natasha Bilkey in the Gilroy Laboratory.

Plasmid Containing Transgenic Insertion (Digestion)

  1. Isolate genomic DNA from WT Col-0
  2. Perform PCR with primers containing restriction enzymes  that select for gene of interest
  3. Perform gel electrophoresis
  4. Place gel in a petri dish
  5. Using UV transilluminator, poke both ends of the desired bands with sterile toothpicks, cut band for gene of interest using razor blade, remove band (image before cutting, after cutting, and again if you needed to cut a 2nd time)
  6. Extract DNA using Promega PCR/Gel clean-up kit

7. Ligate PCR product into pAN19 vector using restriction enzymes (3:1 donor fragment to recipient)

8. Run gel electrophoresis to affirm successful ligation

9. Ligate fragments into ??? vector at a 3:1 donor fragment to recipient (pENTR11) fragment ratio

Transformation with Agrobacterium (Containing Rescue Plasmid)

Plant-dip method with Agrobacterium as soon as the inflorescence stem appears

Detection of Kanamycin Resistance

Kanamycin resistance is included in the vector with the transgenic insertion

Kanamycin resistance SHOULD be connected with proper transgenic insertion

  1. Sow 1000’s of seeds on an agarose plate containing kanamycin (T0)
  2. Select four (alive) seedlings from T0 and grow up to produce seed
  3. Sow 100 seeds of each chosen seedling onto four agarose plates containing kanamycin (T1)
  4. Select for the plate that expresses a 3:1 ratio of kanamycin resistance (25% die)
  5. A plate that shows 100% resistance has the insertion in many places on the chromosome (0% die)
  6. Select six seedlings from the 3:1 plate and grow up to produce seed (T2)
  7. There should be a 2:1 ratio of heterozygous to homozygous kanamycin resistance (two should be homozygous)
  8. Sow 100 seeds for each chosen seedling onto six agarose plates containing kanamycin (T2)
  9. Select for the plate that expresses 0% death
  10. 0% death = homozygous
  11. 25% death = heterozygous
  12. At this point we are sure that seedlings from this plate are 100% homozygous for kanamycin resistance, but because of the rare chance that the transgenic insertion was lost, we must test for the presence of the transgenic insertion via PCR

Detection of Transgenic Insertion

  1. Isolate genomic DNA from seedlings homozygous for kanamycin resistance (Do not use chelex, use typical DNA extraction methods)
  2. Perform PCR using primers for the insert
  3. Perform gel electrophoresis
  4. Confirm homozygosity of transgenic insertion.