https://gcat.davidson.edu/GcatWiki/index.php?action=history&feed=atom&title=Jan_12_Notes 2026-04-15T00:23:09Z Revision history for this page on the wiki MediaWiki 1.28.2 https://gcat.davidson.edu/GcatWiki/index.php?title=Jan_12_Notes&diff=17741&oldid=prev 2016-01-14T18:30:00Z

<p></p> <table class="diff diff-contentalign-left" data-mw="interface"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr style='vertical-align: top;' lang='en'> <td colspan='2' style="background-color: white; color:black; text-align: center;">← Older revision</td> <td colspan='2' style="background-color: white; color:black; text-align: center;">Revision as of 18:30, 14 January 2016</td> </tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l1" >Line 1:</td> <td colspan="2" class="diff-lineno">Line 1:</td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>6 snakes 3 fed/3 unfed</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>6 snakes 3 fed/3 unfed</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Flash-fozen in dry ice and ethanol to preserve RNA</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Flash-fozen in dry ice and ethanol to preserve RNA</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>0.1g samples of frozen tissue were shaved off</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>0.1g samples of frozen tissue were shaved off</div></td></tr> <tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>-possibility for the tissue to not have been representative</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>&#160;</div></td></tr> <tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>-yielded 100 ug RNA</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"> </ins>-possibility for the tissue to not have been representative</div></td></tr> <tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>-need 10 ug for amplification</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"> </ins>-yielded 100 ug RNA</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"> </ins>-need 10 ug for amplification</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>&#160;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Isolate only mRNA to use to create cDNA</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Isolate only mRNA to use to create cDNA</div></td></tr> <tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>-beads used to isolate mRNA were covered in poly-T tails</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"> </ins>-beads used to isolate mRNA were covered in poly-T tails</div></td></tr> <tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>-beads were also magnetic</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"> </ins>-beads were also magnetic</div></td></tr> <tr><td class='diff-marker'>−</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>-isolates 2% of RNA</div></td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins class="diffchange diffchange-inline"> </ins>-isolates 2% of RNA</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>&#160;</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>removed from bead and mixed with Reverse Transcriptase (RT), dNTP's, and made cDNA</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>removed from bead and mixed with Reverse Transcriptase (RT), dNTP's, and made cDNA</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>RNA fragmentation - normally want to avoid fragmenting RNA BUT: High-throughput sequencing only produces 75 bp reads</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>RNA fragmentation - normally want to avoid fragmenting RNA BUT: High-throughput sequencing only produces 75 bp reads</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Fragmentation allows for more coverage in sequencing</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Fragmentation allows for more coverage in sequencing</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>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</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>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</div></td></tr> <tr><td colspan="2">&#160;</td><td class='diff-marker'>+</td><td style="color:black; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;"></ins></div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Purified PCR product was run on a gel. Fragment of about 500 bp was excised and reamplified to confirm data.</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>Purified PCR product was run on a gel. Fragment of about 500 bp was excised and reamplified to confirm data.</div></td></tr> <tr><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>These fragments were then used to produce the RNA seq data.</div></td><td class='diff-marker'>&#160;</td><td style="background-color: #f9f9f9; color: #333333; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #e6e6e6; vertical-align: top; white-space: pre-wrap;"><div>These fragments were then used to produce the RNA seq data.</div></td></tr> </table>

Nielder https://gcat.davidson.edu/GcatWiki/index.php?title=Jan_12_Notes&diff=17740&oldid=prev 2016-01-14T18:29:18Z

<p>Created page with &quot;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...&quot;</p> <p><b>New page</b></p><div>6 snakes 3 fed/3 unfed<br /> Flash-fozen in dry ice and ethanol to preserve RNA<br /> 0.1g samples of frozen tissue were shaved off<br /> -possibility for the tissue to not have been representative<br /> -yielded 100 ug RNA<br /> -need 10 ug for amplification<br /> Isolate only mRNA to use to create cDNA<br /> -beads used to isolate mRNA were covered in poly-T tails<br /> -beads were also magnetic<br /> -isolates 2% of RNA<br /> removed from bead and mixed with Reverse Transcriptase (RT), dNTP's, and made cDNA<br /> RNA fragmentation - normally want to avoid fragmenting RNA BUT: High-throughput sequencing only produces 75 bp reads<br /> Fragmentation allows for more coverage in sequencing<br /> 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<br /> Purified PCR product was run on a gel. Fragment of about 500 bp was excised and reamplified to confirm data.<br /> These fragments were then used to produce the RNA seq data.</div>

Nielder