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 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	–