Micro-CTAB DNA Extraction Procedure for Fresh Material

Overview

Teaching: 15 minutes
Exercises: 2.5 hours

Questions

• How to extract DNA from fresh pulse tissues?

Objectives

• Use CTAB Extraction Buffer to help extract DNA from fresh pulse crops.

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.

The following protocol can be used to extract DNA from all pulse crops:

1. Collect a small coleoptile (2.5 cm) or leaf tissue (~100 mg) sample from the plant. The tissue should be placed in a 1.5 mL micro-centrifuge tube and placed on ice.
2. Grind the plant tissue in the tube using a sterile micro-pestle.
3. Add 500 µl of 65°C 2X CTAB Extraction Buffer. Mix thoroughly by inversion.
4. Incubate tubes in a 65°C water bath for 5-10 minutes.
5. Add 500 µl of chloroform/isoamyl-alcohol (24:1). Gently mix the 2 phases together.
6. Centrifuge the samples @ 13 000 rpm for 10 minutes.
7. Transfer the upper, aqueous phase to a new 1.5 mL micro-centrifuge tube.
8. Add 1/10 volume (~50 µl or 2-3 small drops) of 65ºC 10% CTAB Extraction Buffer. Mix thoroughly by inversion.
9. Incubate tubes in a 65°C water bath for 5-10 minutes.
10. Add 500 µl of chloroform/isoamyl-alcohol (24:1). Gently mix the 2 phases together.
11. Centrifuge the samples @ 13 000 rpm for 10 minutes.
12. Transfer the upper, aqueous phase to a new 1.5 mL micro-centrifuge tube.
13. Add 800 µl of cold 95% ethanol. Mix thoroughly and place @ -20°C 30 minutes.
14. Centrifuge the samples @ 13 000 rpm for 10 minutes.
15. Decant the ethanol and add 500 µl of cold 70% ethanol. Mix thoroughly and place @ -20°C for 5-15 minutes
16. Centrifuge the samples @ 13 000 rpm for 10 minutes.
17. Pipette off the ethanol and let tubes air dry. Alternatively a speed-vac can be used.
18. Resuspend DNA in 100 µl of sterile H₂O.
19. Add 1 µl of 10 mg/mL RNAse A.

Notes:

• Samples can be left overnight after adding the ethanol in step 13 or 15.
• While samples are incubating in the 65ºC H₂O bath, they should periodically be mixed by inversion.
• Can expect an average product of ~200-400 ng/µl (x100µl) = 20 – 40 µg
• Have gotten over 1000 ng/µl = 100 µg.
• Final tubes should be labeled with complete name (for varieties) or population and line number (not entry number).

Key Points

• Read through Safety Information before you start the experiment.

• This protocol can be used to extract DNA from all pulse crops.