final_talk_7_harner_marques_vaden

William Harner, Lily Vaden, Manny Marques
Dr. Laura Lorentzen
NJCSTM Kean University
CONSTRUCTION OF THE
MUSTARD PLANT GAPDH
GENE CONSENSUS
SEQUENCE

Mustard tahtsai
 Food: Spices, greens, condiments
 Medicinal: anti-inflammatory, laxative, pain
relief[1]
 Nutritional: Vitamin A, C, calcium, and
potassium.
GAPDH gene[2]
 Codes for a crucial glycolysis enzyme
 Highly Conserved
 Used as a house keeping gene
MUSTARD GAPDH GENE
1-Annie’s Remedy. 2000-2012. Medicinal Uses and Health benefits of Mustard. [http://www.anniesremedy.com/herb_detail369.php#n59]
Accessed 2012 June 6
2-. Bailey R. 10 Steps of Glycolysis. About.com Biology. [http://biology.about.com/od/cellularprocesses/a/aa082704a.htm]Accessed 2012 June 6.

Helen Angaine
MS Biotechnology student
 Attempted GAPDH gene sequencing
 Reaction failed due to insufficient samples
 Proposed continuation with more samples
Our Work
 Use of Biological systems class’s bacterial clones
 Larger sample- 28 vs. 8
 Similar methodology and analysis
PRIOR WORK

DNA extraction
Amplification
 Initial PCR
 Nested PCR
 Purification
Ligation to pJet plasmid
Transformation and replication
DNA isolation and purification
DNA ISOLATION[3]
3-Bio-Rad Laboratories. 2010 Biotechnology ExplorerTM
Sequencing and Bioinformatics Module Instruction Manual. Bio Rad Laboratories, Inc.

4 different primers
 2 forward, 2 reverse
 Ensures entire GAPDH gene is encoded
Submitted to a gene sequencing facility, Genewiz
SETTING UP SEQUENCING[3]

Genewiz assessed samples for quality control
 No priming
 Early termination
 High background noise
Manually determined viable clones
 Presence of “N” bases
 3 viable preparations
 Q20 Values determined ambiguity of bases
PICKING CLONES TO SEQUENCE

iFinch generated sequence per primer added[4]
iFinch constructed a chromat[4]
 4 chromats combined to create a single contig
IFINCH
4--Huang, X. and Madan, A. (1999) CAP3: A DNA sequence assembly program. Genome Res., 9, 868-877.

Forward GAP primer
 Didn’t prime certain samples
 High background noise
 Early termination
 Addition caused color change- possible contamination/degradation
Reverse GAP primer
 Consistent “N” base sections
 Difficult to compile contig
PROBLEMS ENCOUNTERED

Inconclusive
Not enough viable clones contigs
3 of our prep, 2 from Helen’s prior work
A good consensus requires 6-8
Clones worked fine, duplicated by Dr. Lorentzen
New set of primers or sequencing kit
2nd
attempt on sequencing
Gene may be different from other plants
Vector and primer change
RESULTS AND CONCLUSIONS

final_talk_7_harner_marques_vaden

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