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= N2 adaptor ligation set up = * group sample into 3 different groups based on total completely MmeI digested DNA amount including ** groupA: <= 20ng **20ng < groupB <= 40ng **40ng < groupC <= 60ng == 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. * so for lower amount of MmeI digested DNA, dilute adaptor with different dilution {| {{table}} | align="center" style="background:#f0f0f0;"|'''final conc''' | align="center" style="background:#f0f0f0;"|'''so for lower amount of MmeI digested DNA, dilute adaptor with different dilution''' |- | 4uM||20ng DNA, dilute 5x (2ul of 20uM adaptors with 8ul 1X Stoffel buffer) |- | 8uM||40ng DNA dilute 2.5x (4ul of 20uM adaptors with 6ul 1xStoffel buffer) |- | 12uM||60ng DNA dilute 1.67x (3ul of 20uM adaptors with 2ul 1x Stoffel buffer) |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Group (by DNA amount)''' | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''conc.(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="background:#f0f0f0;"|'''N2 adapters conc (uM)''' | align="center" style="background:#f0f0f0;"|'''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)''' |- | A||P1C||0.56||30.00||16.84||4.00||0.40||8.00||1.00||0.60||39.40 |- | C||P2C||1.60||30.00||47.94||12.00||0.40||8.00||1.00||0.60||39.40 |- | B||P3C||0.97||30.00||28.97||8.00||0.40||8.00||1.00||0.60||39.40 |- | B||P4C||1.33||30.00||39.91||8.00||0.40||8.00||1.00||0.60||39.40 |- | B||P5C||1.42||30.00||42.46||8.00||0.40||8.00||1.00||0.60||39.40 |- | B||P6C||0.99||30.00||29.61||8.00||0.40||8.00||1.00||0.60||39.40 |- | B||P7C1||1.41||30.00||42.35||8.00||0.40||8.00||1.00||0.60||39.40 |- | A||P7C2||0.46||30.00||13.71||4.00||0.40||8.00||1.00||0.60||39.40 |- | A||P8C||0.57||30.00||17.01||4.00||0.40||8.00||1.00||0.60||39.40 |- | A||P1P||0.59||30.00||17.62||4.00||0.40||8.00||1.00||0.60||39.40 |- | B||P2P||0.94||30.00||28.29||8.00||0.40||8.00||1.00||0.60||39.40 |- | A||P3P||0.61||30.00||18.17||4.00||0.40||8.00||1.00||0.60||39.40 |- | C||P4P||1.59||30.00||47.80||12.00||0.40||8.00||1.00||0.60||39.40 |- | B||P5P||0.92||30.00||27.58||8.00||0.40||8.00||1.00||0.60||39.40 |- | A||P6P||0.42||30.00||12.64||4.00||0.40||8.00||1.00||0.60||39.40 |- | B||P7P||1.15||30.00||34.45||8.00||0.40||8.00||1.00||0.60||39.40 |- | B||P8P1||1.29||30.00||38.72||8.00||0.40||8.00||1.00||0.60||39.40 |- | C||P8P2||2.03||30.00||60.95||12.00||0.40||8.00||1.00||0.60||39.40 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Reagents''' | align="center" style="background:#f0f0f0;"|'''1x''' | align="center" style="background:#f0f0f0;"|'''Mix A (6.5 rxn mix)''' | align="center" style="background:#f0f0f0;"|'''Mix B (9.5 rxn mix)''' | align="center" style="background:#f0f0f0;"|'''Mix C (4.5 rxn mix)''' |- | 5xQuickLigase buffer||8.00||52.00||76.00||36.00 |- | QuickLigase||1.00||6.50||9.50||4.50 |- | N2 Adapters, conc. (Gr. A, B or C) ||0.40||2.60||3.80||1.80 |- | H2O||0.60||3.90||5.70||2.70 |- | Total||10.00||65.00||95.00||45.00 |}<br> - incubate at RT for 15 min<br> - purify with Ampure beads, elute with 30ul EB<br>
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