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== BSPP capture set up == * Since I expected that the amount of template would increase after amplification, I roughly estimate it to be ~10X--> after column purification, I assume 5X left over. * I therefor increase the amount of BSPP to 5X based on 1000:1 probe to target ratio. * Perform BSPP capture the same volume and incubation time as experiment on 2014-06-01: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-6-1]] {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Input BIS-DNA (ng) in pre-amp''' | align="center" style="width:120px;background:#f0f0f0;"|'''Assume 5X amount after pre-amp (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Probe required (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''V1N3 conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''V1N3 volume (ul)''' |- | 5||25||1.10||0.73||1.51 |- | 10||50||2.20||0.73||3.01 |- | 25||125||5.50||6.75||0.81 |- | 50||250||11.00||6.75||1.63 |} {| {{table}} border = 1 | align="center" style="width:160px;background:#f0f0f0;"|'''strip 1''' | align="center" style="width:60px;background:#f0f0f0;"|'''5a''' | align="center" style="width:60px;background:#f0f0f0;"|'''5a''' | align="center" style="width:60px;background:#f0f0f0;"|'''10a''' | align="center" style="width:60px;background:#f0f0f0;"|'''10a''' | align="center" style="width:60px;background:#f0f0f0;"|'''25a''' | align="center" style="width:60px;background:#f0f0f0;"|'''25a''' | align="center" style="width:60px;background:#f0f0f0;"|'''50a''' | align="center" style="width:60px;background:#f0f0f0;"|'''50a''' |- | Volume (ul)||5.00||5.00||5.00||5.00||5.00||5.00||5.00||5.00 |- | H2O (ul)||2.49||2.49||0.99||0.99||3.19||3.19||2.37||2.37 |- | V1N3 amount (ng)||1.10||1.10||2.20||2.20||5.50||5.50||11.00||11.00 |- | V1N3 conc. (ng/ul)||0.73||0.73||0.73||0.73||6.75||6.75||6.75||6.75 |- | V1N3 volume (ul)||1.51||1.51||3.01||3.01||0.81||0.81||1.63||1.63 |- | 10X Ampligase Buffer||1.00||1.00||1.00||1.00||1.00||1.00||1.00||1.00 |- | Total||10.00||10.00||10.00||10.00||10.00||10.00||10.00||10.00 |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 1ul SLN mix (2U/ul Hemo Klentaql fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 5h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 2min -> 4C hold.
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