atpF intron 4-1 to 4-12

Steven Jackson and Jenavieve Rummel performed PCR amplification for eleven orchid species. The DNAs used for amplification for each species used were extracted by other university students and professors.

The area applified was the atp-F intron region. Each DNA sample was loaded into a gel along with water as a negative control, and a know genetic marker for base pair length comparison. The gels were imaged using a ultraviolet light camber. The results of the amplification and gel are below. The results were fairly good with all but lane 7 amplifying, and lanes 4 and 13 having weak results.

 

 

 

 

 

 

 

Lane	DNA Code	Species Name	Success
1	Marker
2	Control
3	4.1	Chiloschista segawae	++
4	4.2	Phalaenopsis viridis	+
5	4.3	Rhynchostylis gigantea	++
6	4.4	Holcoglossum subulifolium	++
7	4.5	Phalaenopsis equestris	–
8	4.6	Rhynchostylis coelestis	++
9	4.7	Angraecum didieri	++
10	4.8	Aerangis luteo-alba	++
11	Marker
12	4.9	Cleisostoma schneideri	++
13	4.10	Cleisostoma subulatum	+
14	4.11	Chroniochilus virescens	++

 

PCR results atp-F intron from 4-23 through 4-33

Ashley Lovelace and Nicole Neal performed PCR amplification for eleven different orchid species. The DNA for each species used was extracted by other students.

The purpose was to amplify the plastid-encoded atp-F intron region of the chloroplast DNA. Each DNA sample was loaded into a gel along with water as a negative control. The gels were imaged using ultraviolet light.  The atp-F intron region is 1344bp. We found that are samples were larger than 1000bp using the molecular mass ruler and concluded that they were around the same molecular size as the atp-F intron. Majority of the samples were the same size, however, sample 14 is slightly larger than the other samples.

The results are displayed below:

Figure 1: Gel Electrophoresis Results (AL, NN)

Lane #	DNA Code	Species	Success
1	Molecular Mass Ruler	n/a
2	(-) Control	n/a
3	4-23	Grosourdya appendiculata	++
4	4-24	Smitinandia micrantha	++
5	4-25	Acampe papillosa	++
6	4-26	Schoenorchis fragrans	–
7	4-27	Drymoanthus minutus	++
8	4-28	Polyrrhiza lindenii	++
9	4-29	Gastrochilus fuscopunctatus	++
10	Molecular Mass Ruler	n/a
11	4-30	Diaphananthe pellucida	++
12	4-31	Sarcoglyphis comberi	++
13	4-32	Sarcophyton pachyphyllus	++
14	4-33	Phalaenopsis taenalis	++

atpF intron from 4-12 to 4-22

Sherri Field Gard and Melissa Fry conducted a PCR amplification of extracted orchid DNS’s 4-12 through 4-22 for target region atpF intron. Samples 4-14 and 4-20 both failed to produce a viable yield. All of the other samples produced either a strong or mild success rate.  After comparing our samples with the provided molecular mass ruler, we determined that the majority of our samples measured approximately 1,000 bp.

Based on the plastid gene map of Phalaenopsis aphrodite we were able to identify the location of atpF intron. In relation to the gene map, atpF intron is related to genes that code for subunits of ATP synthase complex.

During gel electrophoresis set up, one of our primers, 4-16, was out of order and subsequently was misplaced in the lane order. As seen on the image below, we moved sample 4-16 to the bottom lane to the far right.

Above: Figure 1

LANE NUMBER	DNA CODE	GENUS SPECIES	PCR RESULTS
GEL MARKER
NEGATIVE CONTROL
1	4-12	Chroniochilus virescens	++
2	4-13	Thrixspermum bromeliforme	++
3	4-14	Holcoglossum flavescens	–
4	4-15	Renanthera storei	+
5	4-16	Trichoglottis amesiana	++
6	4-17	Omoea philippinensis	++
7	4-18	Gastrochilus acutifolius	+
8	4-19	Trichoglottis bipunctata	+
9	4-20	Malleola constricta	–
10	4-21	Phalaenopsis lobbii	++
11	4-22	Armordorum siamense	++

atpF intron PCR Gel for samples 4-34 through 4-44

The PCR reactions was set up by Archana Patel and Emmanuel  Boafo-Asare from extracted Orchid DNA samples 4-34 through 4-44. There were no errors in setting up the reaction. The gel electrophoresis process was set up successfully; the only error in prepping was forgetting to add EZ vision to the marker before loading it into the gel. As a result, the marker was not stained and therefore could not be visualized when the gel was imaged.

All the samples were amplified successfully in the PCR with the exception of Adenoncos Sp. in lane B4. This sample failed to amplify either due to having inadequate amount of template to be amplified or failure to add sufficient amount of enzyme to synthesize new templates. Observing the amplified samples, I would estimate the size of the bands to be around 1100Kb even though the marker is not visible to accurately compare the bands.

Lane Number	DNA Code	Genus Species	PCR Results
A1	4-34	Polystachya longiscapa	++
A2	4-35	Cyrtorchis monteirae	++
A3	4-36	Tridactyle tridentata	++
A4	4-37	Jumellea comorensis	++
A5	4-38	Aerangis biloba	++
A6	4-39	Phalaenopsis stuartiana	++
A7	marker
A8	empty
A9	empty
A10	empty
B1	4-40	Phalaenopsis amboinensis	++
B2	4-41	Phalaenopsis gigantea	++
B3	4-42	Paraphalaenopsis serpentelingua	++
B4	4-43	Adenoncos sp.	—
B5	4-44	Papilionanthe pedunculata	++
B6	NC	water
B7	marker
B8	empty
B9	empty
B10	empty