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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-6 == Capture set up (Matt's project) == * Set up capture in 30ul NTC (no targets) 1-1. gDNA (12878) 2-1. Total RNA 3-1. ds cDNA no RNasA treatment 4-1. ds cDNA RNaseA treatment 1-2. gDNA (12878) 2-2. Total RNA 3-2. ds cDNA no RNasA treatment 4-2. ds cDNA RNaseA treatment == Probe calculation == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:target''' | align="center" style="background:#f0f0f0;"|'''1000:1''' | align="center" style="background:#f0f0f0;"|'''''' |- | Probe size||12,335||probes |- | Template||300||ng |- | Human gDNA MW||1.9500E+12||g/mol (3E+09 * 650Da/bp+157.9Da) |- | Human gDNA (300ng)||1.5385E-19||mol (300ng/1.9500E+12 g/mol) |- | Probe required (1000:1)||1.5385E-16||mol (1.5385E-19mol x 1000) |- | Probe MW (12,335 probes, 170nt)||6.8151E+08||g/mol (12335 probes * 170nt * 325Da/nt + 79Da) |- | Amount probe required||1.05E-07||g (6.8151E+08 g/mol * 1.5385E-16mol) |- | Amount probe required||105.00||ng |} * Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul * Note: ** I showed Matt how to calculate probes and he did calculation, and the number he got agreed with this calculation **Even the target amounts in each reaction are not the same, I used the same probe amount in every reaction based on gDNA calculation == Probes and AmpLigase Buffer Mix == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''9.5 rxn mix''' |- | 12K Insitu probes (22ng/ul)||4.77||45.32 |- | 10X AmpLigase buffer||3.00||28.50 |- | Total||7.77||73.82 |} {| class="wikitable" style="text-align:center;{{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''12K Insitu probes''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase buffer''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Targets (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount <br>in capture (ng)''' |- | NTC||4.77||3.00||0||0||22.23||30.00||0.00 |- | 1-1. gDNA (12878)||4.77||3.00||45.10||6.65||15.58||30.00||299.92 |- | 2-1. Total RNA||4.77||3.00||1000.00||1.00||21.23||30.00||1000.00 |- | 3-1. ds cDNA no RNasA treatment||4.77||3.00||15.20||19.00||3.23||30.00||288.80 |- | 4-1. ds cDNA RNaseA treatment||4.77||3.00||5.84||19.00||3.23||30.00||110.96 |- | 1-2. gDNA (12878)||4.77||3.00||45.10||6.65||15.58||30.00||299.92 |- | 2-2. Total RNA||4.77||3.00||1000.00||1.00||21.23||30.00||1000.00 |- | 3-2. ds cDNA no RNasA treatment||4.77||3.00||10.20||19.00||3.23||30.00||193.80 |- | 4-2. ds cDNA RNaseA treatment||4.77||3.00||7.77||19.00||3.23||30.00||147.63 |} '''Program'''<br> * 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h * -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) * -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. == AmpLigase enzyme mix == {| class="wikitable" style="text-align:center;{{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume 30ul''' |- | AmpLigase||5||U/ul||0.5||U/ul||3.00 |- | 10x AmpLigase Buffer||10||x||1||x||3.00 |- | H2O||||||||||24.00 |- | Total||||||||||30.00 |} * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-14
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