Protocol for plant DNA extraction and PCR

Read completely the first time through!

1. Add small amount² of leaf tissue to 1.5 mL microcentrifuge tube.

2. Add 180 µL TE Buffer³

3. Add 20 µL DNA extraction buffer⁴

4. Grind thoroughly with blue Kontes pestle but leave small bits of leaf tissue visible (use a clean pestle for each sample)

5. Add 800 µL sterile H₂0⁵; Vortex 30 seconds

Procedure template for your eLab report

√ each sample	procedure
	+ small amount of leaf tissue to 1.5mL tube
	+ 180 uL TE Buffer
	+ 20 uL DNA extraction buffer
	Grind, leaving small bits visible
	+ 800 uL sterile H20

6. Add 1 µL of above DNA solution to the following in a PCR tube on ice:

12.5 µL 2x Taq mix

1.0 µL of forward primer (10 pm/µL working stock)

1.0 µL of reverse primer (10 pm/µL working stock)

9.0 µL nuclease-free H₂0

0.5 µL MgCl⁶ (25 mM stock)

If performing PCR on more than 4 samples, it is usually best to make a cocktail that includes 1 extra reaction. Example: 8 DNA samples (including a wildtype) to test + 1 negative (no DNA) control + 1 positive (dilute plasmid stock with gene of interest on it) control + 1 extra reaction = cocktail for 11 samples

Preparing PCR cocktail template for eLab report

√	uL	#	Final amount
__	+ 12.5 x __ = ___ uL 2x Taq mix
__	+ 1.0 x __ = ___ uL forward primer (10pm/ul)
__	+ 1.0 x __ = ___ uL reverse primer (10pm/ul)
__	+ 9.0 x __ = ___ uL sterile H20
__	+ 0.5 x __ = ___ uL MgCl (25 mM stock)

7. Run PCR; 35 cycles has given good results with this extraction procedure.

Initial melt	94 C
Melt	94 C
Anneal	45-65 depending on Tm of primers or experience
Elongation	72 C
Cycle repeat	34 times
Final extension	10 minutes at 72 C

^1- This procedure is a modified version of the one published by Ichiro Kasajima, et al.

Kasajima, I., Ide, Y., Ohkama-Ohstsu, N., Hayashi, H., Yoneyama, T., and Fujiwara, T. (2004) Plant Molecular Biology Reporter 22, 49-52.

^2- Tissue should be the size of a small cotyledon on a young plant: tissue should be about this size or smaller: O

^3- 10 mM Tris-HCl (pH 8) and 1 mM ethylenediaminetetraacetic acid (EDTA).

^4- 200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, and 0.5% SDS.

^5- This 5x dilution may lower the amount of suspected polymerase inhibitors in the sample. Whatever the mechanism, this step is necessary; results were only consistent when a dilution was used.

^6- This brings the total amount of MgCl to 2mM and seems to present the bands in a more uniform fashion. Because the addition of less than a µL is inaccurate using our pipets, a cocktail should be used: if the number of samples makes a cocktail redundant than a dilution of MgCl should be made up.

Protocol designed by Michael Baughan, Salisbury University, Jan. 2007