DNA Harvest and PCR amplification

Purpose: the purpose of this experiment was to harvest DNA from previously selected orchid species and amplify regions, using PCR method, of their DNA that contain atpF genes.

PCR results

Results: DNA regions from each subject were successfully amplified, as shown in the electrophoresis gel image above.

Lane #	Genus species	PCR results
1	DNA ladder	n/a
2	Phalaenopsis tetraspsis	+
3	Phalaenopsis tetraspsis	+
4	Schoenorchis juncifolia	+
5	Schoenorchis juncifolia	+
6	Robiquetia spathulata	+
7	Robiquetia spathulata	+
8	Pomatocalpa wendlandorum	+
9	Pomatocalpa wendlandorum	+
10	negative control

PCR atpF intron

Jamie Cone and Faye Montgomery extracted DNA from two plant species, Phalenopsis tetrapsis and Schoenorchis juncifola and the prepared for PCR extraction.  The following week, two previously extracted samples, Robiquetia spathulata and Pomatocalpa wendlandorum were also prepared.  The goal of this reaction was to target atpF intron, a non coding region of our orchid chloroplast DNA, and amplify it.  These samples were then loaded for gel electrophoresis.

Species	10 ng/ml
Phalenopsis tetrapsis	60.5
Schoenorchis juncifolia	16.3
Robiquetia spathulata	138.4
Pomatocalpa wendlandorum	84.8

Lane	DNA code	Species/Marker	Success
1	Molecular Mass Ruler	n/a
2	3-1	Phalenopsis tetrapsis	++
3	3-1	Phalenopsis tetrapsis	++
4	3-6	Schoenorchis juncifola	+
5	3-6	Schoenorchis juncifola	+
6	272	Robiquetia spathulata	++
7	272	Robiquetia spathulata	++
8	278	Pomatocalpa wendlandorum	++
9	278	Pomatocalpa wendlandorum	++
10	marker	n/a

PCR atpF

David Smith and Jacob F. extracted DNA from Micropera pallida. We were then given 3 other samples: Pomatocalpa bicolor, Stereochilus hirtus, and Luisa filiformis. We amplified the DNA samples of all four species to run gel electrophoresis.

Gel electrophoresis of DNA samples

 

 

 

 

 

 

Lane	DNA Code	Species or Marker	Success
1	n/a	Molecular mass ruler	n/a
2	n/a	Negative control	n/a
3	3-5	Micropera pallida	+
4	3-5	Micropera pallida	+
5	3-9	Pomatacalpa bicolor	++
6	3-9	Pomatacalpa bicolor	++
7	382	Stereochilus hirtus	++
8	382	Stereochilus hirtus	++
9	384	Luisa filiformis	++
10	384	Luisa filiformis	++

PCR atp-F

On September 30th, 2011, Robert Lavender and Ashley Orr extracted DNA from two Orchid species, Plectrelminthus caudatus and Polystachya zambesiaca.  On October 6th, 2011, these and two previously extracted DNA samples, Robiquetia merrillii and Stereochilus hirtus, were prepared for a PCR reaction.  The goal of this PCR reaction was to amplify the atp-F region of the chloroplast DNA of our orchids. Two DNA samples for each of the four orchid species were set up, as well as a negative control (water). These samples were then loaded into a gel using the reaction mixture and loading dye to prepare them for gel electrophoresis. After the gels were run, they were imaged using ultraviolet light.

All of our reactions were highly successful with the exception of our last two lanes, both originating from Stereochilus hirtus.  This may have been due to the age of the DNA sample.  Both qualified and quantified data are displayed below.

Gel with DNAs 3-4, 3-8, 366, and 376

Lane	DNA Code	Speices/ Marker	Success
1	–	Molecular Mass Ruler	N/A
2	–	Negative Control (Water)	N/A
3	3-4	Plectrelminthus caudutus	++
4	3-4	Plectrelminthus caudutus	++
5	3-8	Polystachya zambesiaca	++
6	3-8	Polystachya zambesiaca	++
7	366	Robiquetia merrillii	++
8	366	Robiquetia merrillii	++
9	376	Stereochilus hirtus	–
10	376	Stereochilus hirtus	–

Species	ng/μl
Plectrelminthus caudatus	47.7
Polystachya zambesiaca	76.4
Robiquetia merrillii	81.2
Stereochilus hirtus	null

*This post was updated November 4th, 2011 by Ashley Orr

*Updated 12/13/2011

PCR atp-F

 

Kelley Stokes and Harsh Patel extracted DNA from Sedirea japonica and were given DNA from three other species on which to run a PCR reaction. Lanes 3 – 5 and 7 – 10 were very successful. Lane 6 was weak, but also successful. Our yields were: Sedirea japonica – 59.0 ng/ul, Podangis dactyloceras – 84.4 ng/ul, Cleisostoma scolopendrifolium – 76.5 ng/ul, and Eurychone rothschildiana – 50.6 ng/ul.

Lane	DNA Code	Species or Marker	Success
1	n/a	Molecular mass ruler	n/a
2	n/a	Negative control	n/a
3	3-2	Sedirea japonica	++
4	3-2	Sedirea japonica	++
5	3-10	Podangis dactyloceras	++
6	3-10	Podangis dactyloceras	++
7	387	Cleisostoma scolopendrifolium	++
8	387	Cleisostoma scolopendrifolium	++
9	389	Eurychone rothschildiana	++
10	389	Eurychone rothschildiana	++

PCR atpF

1% agarose gel with DNAs 3-3, 3-7, 300 and 364

PCR amplification of chloroplast region atpF for four different orchid species was obtained on September 30 by Alivia McMahan and James Murphy. Agarose gel electrophoresis was then performed to reveal qualitative knowledge of the results. This method revealed a variety of success rates concerning the yields of the extracted DNA from the four orchid species.

Quantification of orchid 3-7 (lanes 5&6) showed the highest success rate, attributable to the presence of a single band of sufficient size and a yield of 91.7 ng/ul. Orchids 3-3 (shown in lanes 3&4) and 300 (lanes 7&8) were termed mildly successful due to the presence of faint banding patterns. DNA extracted from orchid 3-3 achieved a yield of 7.3 ng/ul where as orchid 300 had a yield of 5.1 ng/ul. Lastly, orchid 364 (lanes 9&10) was termed unsuccessful.

Lane	DNA Code	Species or Marker	Success?
1	N/A	Molecular mass ruler	N/A
2	N/A	Negative control	N/A
3	3-3	Cleisostoma linearilabatum	+
4	3-3	Cleisostoma linearilabatum	+
5	3-7	Holcoglossum wangii	++
6	3-7	Holcoglossum wangii	++
7	300	Cleisostoma teres	+
8	300	Cleisostoma teres	+
9	364	Papilionanthe vandarum	–
10	364	Papilionanthe vandarum	–