
Overview
Kit contains sufficient materials for 12 student workstations, or 96 sequencing reactions. | |
pJETF primer | 1 |
pJETR primer | 1 |
GAPSEQF primer | 1 |
GAPSEQR primer | 1 |
Control plasmid | 1 |
Barcoded 96-well plate | 1 |
Sealing film | 1 |
Colored micro test tubes, 2 ml | 60 |
Curriculum, including Teacher's Guide, Student Manual, and graphic Quick Guide | 1 |
Required Accessories Not Included in Kit: | |
Adjustable micropipets | |
2–20 µl | |
Pipet tips | |
Computers and internet access |
Sequencing and Bioinformatics: part of the Cloning and Sequencing Explorer Series
Sequence and analyze your cloned DNA. Students combine plasmid DNA containing PCR products with sequencing primers in a 96-well plate and send them to a sequencing service.
The portion of the GAPDH gene to be cloned using the Cloning and Sequencing Explorer Series can vary from 0.6 to over 2 kb in length depending on the plant source. To ensure complete coverage of the gene, four sequencing primers are provided: one forward and one reverse primer specific to either side of the pJET1.2 cloning site, and one forward and one reverse primer homologous to different regions in the middle of the GAPDH gene. Thus, four independent sequences will be generated for each plasmid — these sequences can be assembled into a contig using the bioinformatics module. We recommend each student team submit two plasmids for sequencing with all four primers. The ultimate goal of the Cloning and Sequencing Explorer Series is to generate sequence data that can be uploaded into NCBI's GenBank database to be used by other scientists. It is important to generate many sequence-reads to guard against sequencing errors and ensure data accuracy. If multiple groups within a class independently clone the same GAPDH gene, the repetition will be sufficient to confirm the sequence. Additionally, sequencing primers are provided to allow for sequence reads in both forward and reverse directions, providing another method of sequence confirmation.
Once the plasmid has been cloned and sequenced, the real work begins — interpreting the sequence data. This module teaches students the essential bioinformatics skills needed to study biology with the power of computer analysis. The Sequencing and Bioinformatics Module includes a 3-month subscription to the Geneious software, a program that provides students with access to the latest tools in bioinformatics data analysis. After downloading Geneious, raw sequence files are imported and are immediately ready for exploration. Students will view and edit the original chromatograms of their sequences, screen out vector sequences, confirm their sequences are GAPDH, perform NCBI BLAST searches on their data to identify and find related sequences, assemble their forward and reverse sequences into contigs, predict intron and exon structure, transcribe and translate the GAPDH sequence, and compare it to known gene, mRNA, and protein sequences. Finally, confirmed sequences can be added to NCBI’s GenBank database where scientists around the world will be able to use that data for their research.
The sequencing and bioinformatics module is used to analyze a portion of the GAPC gene isolated from a plant. Students are guided through a sequence analysis that allows them to make a real contribution to science!
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