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== PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind1-8) == * I did PCR by adding 10ul of circularized DNA in total 50ul. I used smaller volume because the template amount should be less than the circularized DNA from 330k probe set and I actually did PCR test before (add 2ul of circularized DNA in total 25ul, and the curve shoot up at approx. cycle 14) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''8.5x rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||1.00||8.50 |- | 10uM AmpR6.3Indx (X=1-8)||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||212.50 |- | H2O||13.00||110.50 |} * Aliquot 39ul, add 1ul of AmpR6.3Indx, 10ul of circularized DNA '''Program (Eppendorf Realplex)''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min '''qPCR curve''' [[File:PCRcurve_BlueprintAmp.png| 700px]] * I did the second qPCR by using the same condition as above and got the same result. * Purified all PCR products with 0.8 vol of AmPure beads (I diluted AMPure bead 1:1 ratio with 20%PEG/2.5M NaCl) * Eluted with 30ul H2O * Performed PAGE quantification by loading sample 3ul (1/10 of total purified PCR product). I need to load large volume to make sure I would see the band for PAGE quantification for library pooling. [[File:ZhangLab_2 2013-06-11 18hr 21min_beadpurified_BLUEPRINT.jpg| 500px]] * From the results above, I saw multiple bands from amplification, including ~200bp, 350bp, 400bp and a smear above 400bp band up to almost 500bp. * This result was different from the DMR330k probe capture (~375bp, gap size 180bp). Based on the probe design for this probe set with gap size between 200-280bp, I expected to get the fragments size ~ 400-480bp). * The smaller sizes ~200bp and ~350 should not be the expected product since the size seemed to be too small. I always saw very weak signal of these two bands in DMR330k probe set, so they should be the background. * The result clearly showed that increasing probe:target ratio significantly increased the capture efficiency. I should have tried 1000:1 ratio in this experiment. However, I believe to get higher efficiency when using 1000:1 ratio. I might use 1000:1 ratio for the real experiment. * Increasing template amount also increasing capture efficiency as well, but 50ng template may be too low for capture. Sequencing result should tell the quality of data and target coverage by varying these two factor. * There might be something wrong for 300ng , 500:1 sample since the amplification was poor and PAGE analysis agreed with qPCR curve. I suspected that SLN mix may not delivered right to the whole reaction since the tube of this samples was on the innermost of thermocycle and I couldn't add the SLN mix properly because of the lid. Next time, I should be more careful when add any reagent to the reaction. ''' 2013-06-13 ''' * I did PAGE quauntification on the fragments size 400-500bp. Since some samples have very low concentration, I combined them into two pools and did PAGE size-selection ** NP-BSPP-BP-TEST1-1-June6, 2013 Index 1,2,3,5,8 ** NP-BSPP-BP-TEST1-2-June6, 2013 Index 4,6,7 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample #''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 27ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Pool1 volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Pool2 volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' |- | BP-test # 1||AmpR6.3Ind1||0.03||0.83||27.00||NA||0.83 |- | BP-test # 2||AmpR6.3Ind2||0.22||5.91||27.00||NA||5.91 |- | BP-test # 3||AmpR6.3Ind3||0.75||20.36||19.89||NA||15.00 |- | BP-test # 4||AmpR6.3Ind4||1.87||50.40||NA||26.78||50.00 |- | BP-test # 5||AmpR6.3Ind5||1.21||32.76||12.36||NA||15.00 |- | BP-test # 6||AmpR6.3Ind6||2.88||77.65||NA||17.39||50.00 |- | BP-test # 7||AmpR6.3Ind7||2.33||62.86||NA||21.47||50.00 |- | BP-test # 8||AmpR6.3Ind8||0.57||15.44||27.00||||15.44 |} ''' PAGE size-selection '''<br> [[File:ZhangLab_2 2013-06-13 12hr 23min_PAGE-SS.jpg| 350px]] * Each pool was purified in 2-well of 6% 5-well TBE gel * Resuspended pool1 with 10ul H2O * Resuspended pool2 with 15ul H2O * Alan will do qPCR to quantify concentration ''' MiSeq: MiseqJun18: PE, 2x250bp ''' <br>
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