Crude DNA Isolation from Seeds

Overview

Teaching: 10 minutes
Exercises: 30 minutes

Questions

• How to crude DNA Isolation from seeds?

Objectives

• Extract PCR-only quality DNA from a large number of samples.

SAFETY INFORMATION

Before you start, look up the MSDS sheets for all the chemicals used in this protocol. Ensure you follow any recommendations/rules regarding wearing personal protective equipment, emergency procedures, and clean-up/disposal procedures. If you have any questions contact your supervisor or lab manager.

This protocol provides a quick means to extract PCR-only quality DNA from a large number of samples.

Supplies needed:

• Tweezers
• Toothpicks
• Sterile Water
• 96-well PCR micro-plate
• Heating block set to 95°C
• 0.5M Tris-HCL (pH 8.0)
• 0.25M NaOH

Protocol:

Photo of a bean with the seed coat scraped away on the front side.There is a small amount of cotyledon tissue on the tweezers to demonstrate the amount you need for extraction. Try to keep this amount consistent between samples. Ensure you only take a sample of the cotyledon and do not damage the embryo.

1. Using Tweezers (or a Razor Blade or Scalpel – whichever you find easiest), chip off a small area of seed coat to expose the cotyledons.
   • For beans and lentils, seed coat tissue is derived from maternal parent and does not represent genotype of the embryo. Therefore avoid contaminating sample with seed coat tissue.
   • Avoid damaging the embryo when chipping the seed coat.
2. Place a 96-well PCR micro-plate on ice. Add 2 µl of sterile water to each well.
3. Add a small 1-2 µl drop of water to the exposed section of the cotyledon. It is typically best to line up a series of seeds at once.
4. Using a clean toothpick scrape a small amount of seed flour. Transfer the collected sample into the PCR well by dipping and gently swirling the toothpick into the water at the bottom of the sample well.
   • Only a small amount of flour is needed. More is NOT better.
   • Be as consistent as possible with regards to the amount of tissue being collected.
5. Repeat Steps 3-5 until all samples are collected.
6. Once all samples are collected add 10 µl of 0.25M NaOH to each well.
7. Seal samples. Shake/Vortex briefly on plate-mixer if available. Place plate on 95°C heating block for ~2 minutes. Remove and leave at room temperature.
8. Carefully remove seal and add 15 µl of 0.5M Tris-HCl (pH 8.0) to each well.
9. Seal the samples. Shake/vortex on plate-mixer if available. Place plate on 95°C heating block for ~2 minutes. Remove and place on ice.
10. Samples can be used immediately, stored in the fridge for a few days, or frozen indefinitely.
11. For regular PCR, use 1.0-2.0 µl of sample in a 15-25 µl PCR reaction. For KASP-based PCR, dilute the DNA by adding 200 µl of sterile water. Then use 2.0 µl of diluted DNA in a 8-10 µl KASP reaction.

Important Notes:

• For best results, try to be as consistent as possible with the amount of tissue collected. As with any PCR, KASP-based PCR works best when starting samples have same amounts of DNA. By being consistent, you should not have to quantify the DNA.

Key Points

• Read through Safety Information before you start the experiment.

• Avoid contaminating sample with seed coat tissue when handling beans and lentils.

• Avoid damaging the embryo when chipping the seed coat.

• Small amount of seed flour is good enough; more is not better.

• Try to be as consistent as possible with the amount of tissue collected.