Plant Biotechnology -Botany 415/515

PCR Amplification of DNA

1. Thaw all components (except Taq Polymerase) on ice 30 min before the lab.

2. Combine the following components for each reaction (on ice) in an Eppendorf tube:

Tube (1.5 ml Eppendorf)	Distilled water	10X PCR buffer	dNTP	5 unit/µl Taq Polymerase	Total
Wizard Mix	284 µl	36 µl	36 µl	4 µl (ask TA to pipet enzyme)	360 µl

3. Vortex and centrifuge for 30 seconds at 13,000rpm.

4. Combine the following for each reaction in a PCR tube(50 ul total reaction volume).

Tube	Wizard Mix	DDH₂0	A plasmid containing hsp16.6 gene	Cyanobacterium genomic DNA	A plasmid containing cox II gene	Onion genomic DNA	Primers (F and R) for hsp16.6 gene	Primers (F and R) for Cox II gene
1 (Negative control)	47 µl	1 µl					1 µl primer F_h 1 µl primer R_h
2 hsp16.6 gene control	47 µl		1 µl				1 µl primer F_h 1 µl primer R_h
3 hsp16.6 gene	47 µl			1 µl			1 µl primer F_h 1 µl primer R_h
4 (Negative control)	47 µl	1 µl						1 µl primer F_c 1 µl primer R_c
5 cox gene control	47 µl				1 µl			1 µl primer F_c 1 µl primer R_c
6 cox gene	47 µl					1 µl		1 µl primer F_c1 µl primer R_c
7 Positive control	47 µl		PUC18 plasmid provided in the kit 1 ml			PUC18 primers provided in the kit 1 ul F +1 ul R = 2 µl total

. Run the following program (about 2.5 h):

Initial Step

Next 30 cycles:

94 ^oC 1 min
58 ^oC 1 min (or annealing temperature appropriate for particular primer pair)
72 ^oC 1 min (if product is <500 bp), 3 min (if product is >500 bp) for 30 cycles.

Program a final extension at

5. Take 10 µl of PCR product from each tube for gel electrophoresis (Refer to the protocol for agarose gel electrophoresis).

Materials:

sterile water

10X amplification buffer with 15mM MgCl2

10 mM dNTP

50 µM oligonucleotide primer F (forward)

50 µM oligonucleotide primer R (reverse)

5 unit/µl Taq Polymerase

template DNA (1 µg genomic DNA, 0.1-1 ng plasmid DNA) in ddH₂0

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Plant Biotechnology by: