Creation of a Rescue Line

ASOP-0009 Version 1

Table of Contents

1.0: Purpose

The purpose of this SOP is to provide a general transgenic cloning strategy for Arabidopsis thaliana to create a line for mutant rescue.

Note: This is an advanced cloning strategy that requires more scientific context than this method describes.  Previous cloning experience with a molecular biology background is highly recommended.

2.0: Safety

Standard laboratory personal protective equipment (PPE) should be worn when executing this technique.  Refer to Material Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

3.0: Definitions and Acronyms

  • ASOP: Astrobotany Standard Operating Procedure
  • A. thaliana: Shorthand for Arabidopsis thaliana
  • gDNA: Genomic DNA
  • PCR: Polymerase Chain Reaction

4.0: Materials

  • Petri/cell culture dishes
  • Associated Gel Electrophoresis Materials
  • Plating Materials

5.0: Equipment

6.0: Procedure for Plasmid Containing Transgenic Insertion (Digestion)

  1. Isolate the genomic DNA (gDNA) from Arabidopsis thaliana WT Col-0.
  2. Perform Polymerase Chain Reaction (PCR) with primers containing the restriction enzymes that select for the gene of interest
  3. Perform gel electrophoresis.
  4. Place the gel in a petri dish.
  5. Using a UV transilluminator, poke both ends of the desired bands with sterile toothpicks, carefully cut the band for the gene of interest using a razor blade, then carefully remove the band.
  6. Extract the DNA using a Promega PCR/Gel clean-up kit:

  • 250 μL Agarose Degrading Buffer (ABD) [Put at 60°C for 5-10 minutes].
  • 2 x 300 μL Washing Buffer.
  • 25 μL DNA Elution Buffer.
  • Check the concentration to ensure it is at least 10 ng/μL.

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

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

8. Run gel electrophoresis to affirm successful ligation.

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

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

7.0: Procedure for Transformation with Agrobacterium (Containing Rescue Plasmid)

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

  • As the plant grows, the hypocotyl stem would be chimeric as there is no way to differentiate 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.

8.0: Detection of Kanamycin Resistance

NOTE: Kanamycin resistance is included in the vector with the transgenic insertion so kanamycin resistance SHOULD be connected with proper transgenic insertion.

  1. Sow thousands of seeds on an agarose plate containing kanamycin (T0).
  2. Select four (alive) seedlings from T0 and grow them up to produce seeds.
  3. Sow hundreds of 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 seeds (T2).
  7. There should be a 2:1 ratio of heterozygous to homozygous kanamycin resistance (two should be homozygous).
  8. Sow hundreds of 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, there is near certainty that the seedlings from this plate are 100% homozygous for kanamycin resistance, but because of the rare chance that the transgenic insertion was lost, the presence of the transgenic insertion must be tested via PCR.

9.0: 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.

10.0: References

This protocol is attributed to the Gilroy Lab at the University of Wisconsin-Madison.  

Version History

  • Version 1 | Description: ASOP-0009 page created. | Effective: 31Jul2023
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