Difference between revisions of "Jan 12"
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0.1g samples of frozen tissue were shaved off | 0.1g samples of frozen tissue were shaved off | ||
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yielded 100 ug RNA | yielded 100 ug RNA | ||
need 10 ug for amplification | need 10 ug for amplification | ||
Revision as of 19:53, 12 January 2016
6 snakes 3 fed/3 unfed
Flash-fozen in dry ice and ethanol to preserve RNA
0.1g samples of frozen tissue were shaved off
possibility for the tissue to not have been representative
yielded 100 ug RNA
need 10 ug for amplification
Isolate only mRNA to use to create cDNA
beads used to isolate mRNA were covered in poly-T tails
beads were also magnetic
isolates 2% of RNA
removed from bead and mixed with Reverse Transcriptase (RT), dNTP's, and made cDNA
RNA fragmentation - normally want to avoid fragmenting RNA BUT: High-throughput sequencing only produces 75 bp reads
Fragmentation allows for more coverage in sequencing
Now need to prime RNA for sequencing BUT every the RNA has been fragmented Create a library of primers that contain bar code regions (3 nucleotides) and randomly generated hexameters
Purified PCR product was run on a gel. Fragment of about 500 bp was excised and reamplified to confirm data.
These fragments were then used to produce the RNA seq data.
