Difference between revisions of "Jan 12 Notes"
(Created page with "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 representa...") |
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6 snakes 3 fed/3 unfed | 6 snakes 3 fed/3 unfed | ||
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Flash-fozen in dry ice and ethanol to preserve RNA | Flash-fozen in dry ice and ethanol to preserve RNA | ||
0.1g samples of frozen tissue were shaved off | 0.1g samples of frozen tissue were shaved off | ||
| − | -possibility for the tissue to not have been representative | + | |
| − | -yielded 100 ug RNA | + | -possibility for the tissue to not have been representative |
| − | -need 10 ug for amplification | + | -yielded 100 ug RNA |
| + | -need 10 ug for amplification | ||
| + | |||
Isolate only mRNA to use to create cDNA | Isolate only mRNA to use to create cDNA | ||
| − | -beads used to isolate mRNA were covered in poly-T tails | + | -beads used to isolate mRNA were covered in poly-T tails |
| − | -beads were also magnetic | + | -beads were also magnetic |
| − | -isolates 2% of RNA | + | -isolates 2% of RNA |
| + | |||
removed from bead and mixed with Reverse Transcriptase (RT), dNTP's, and made cDNA | 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 | RNA fragmentation - normally want to avoid fragmenting RNA BUT: High-throughput sequencing only produces 75 bp reads | ||
Fragmentation allows for more coverage in sequencing | 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 | 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. | 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. | These fragments were then used to produce the RNA seq data. | ||
Latest revision as of 18:30, 14 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.
