creation of a rescue line

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
  • 250 uL Agarose Degrading Buffer (ABD) [Put at 60°C for 5-10 minutes]
  • 2 x 300 uL Washing Buffer
  • 25 uL DNA elusion buffer
  • Check concentration is at least 10 ng/uL

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

  • 3 ng DNA for every 1 ng vector
  • pAN19 is 270 ng/uL concentration

8. Run gel electrophoresis to affirm successful ligation

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

  • Cut at restriction sites, run PCR to detect proper insertion into vector

Transformation with Agrobacterium (Containing Rescue Plasmid)

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

  • Essentially, as the plant grows, the hypocotyl stem would be chimeric as we don’t know which cells have been transformed and which haven’t
  • We don’t know which ovules or what pollen has been transformed
  • Transgenic insertion may be in one place, in many places all over the chromosome, or not at all

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.