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= N2 adaptor ligation set up = == Adaptor preparation == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc. (uM)''' |- | 100uM PE_N2_adaptor ||2.00||20.00 |- | 100uM PE_b_A ||2.00||20.00 |- | 10x Stoffel buffer ||1.00||1x |- | H2O ||5.00|| |- | Total||10.00|| |}<br> '''Program''' 94C 2min -> 0.2C/sec to 20C -> 4C hold<br> Adapters to ligated product ratio: 10:1<br> Est length of digested products: 212bp (after MmeI)<br> MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole<br> For 100 ng digested product = 100ng / 128,927g/mole = 7.76E-4 nmole * 10:1 = 0.008 nmole adapters required.<br> 20uM Adapters (ul) = 0.008nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.008nmoles/ (20E-3 nmoles/ul) = 0.40 ul <br> * 0.40 ul of 20uM adapters per 100ng digested products {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''conc.(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul) for 100ng''' | align="center" style="background:#f0f0f0;"|'''20uM N2 adaptors volume (ul)''' | align="center" style="background:#f0f0f0;"|'''5xQuickLigase buffer''' | align="center" style="background:#f0f0f0;"|'''QuickLigase (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' |- | 1. HGPS||4.27||23.43||0.40||8.00||2.00||6.17||40.00 |- | 2. cHGPS1||5.46||18.30||0.40||8.00||2.00||11.30||40.00 |- | 3. cHGPS40||4.95||20.21||0.40||8.00||2.00||9.39||40.00 |- | 4. AWS||5.55||18.01||0.40||8.00||2.00||11.59||40.00 |- | 5. cAWS1||5.28||18.95||0.40||8.00||2.00||10.65||40.00 |- | 6. NTC||NA||0.00||0.40||8.00||2.00||29.60||40.00 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Reagents''' | align="center" style="background:#f0f0f0;"|'''1x''' | align="center" style="background:#f0f0f0;"|'''6.5 rxn mix''' |- | 5xQuickLigase buffer||8.00||52.00 |- | QuickLigase||2.00||13.00 |- | N2 Adapters, conc. (Gr. A, B or C) ||0.40||2.60 |- | Total||10.40||aliquot 10.4ul, <br>add MmeI digested DNA, <br>adjust volume with H2O |}<br> * incubate at RT for 15 min<br> * purify with 1 vol. Ampure beads, elute with 30ul EB<br>
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