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= BSPP capture of amplified DNA from Illumina = '''Sample list:''' * 13 samples received on 2014-03-18 [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-18#BSC_samples_from_Illumina]]. Note: I processed only A1-A10 samples which are amplified DNA samples. * 8 samples received on 2014-04-14[[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-4-14#Amplified_DNA_samples_form_Illumina]] ---- ==== 2014-04-07 ==== == BSPP capture round1 using Polymerase from Illumina == * 2014-05-05: Received ~30ul of polymerase with property similar to Amplitaq Stoffel fragment. Conc. 3U/ul. She is not allowed to provide any further info. of this enzyme. * I performed BSPP capture on samples A1-A10 and sample #1-#8 {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Tube ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 200/300ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Tube ID''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''GP1V4 probe''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | align="center" style="background:#f0f0f0;"|'''Total (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' |- | A1||189.77||1.58||A1||14.16||2.26||2.00||20.00||300.00 |- | A2||192.53||1.56||A2||14.18||2.26||2.00||20.00||300.00 |- | A3||142.63||2.10||A3||13.64||2.26||2.00||20.00||300.00 |- | A4||145.67||2.06||A4||13.68||2.26||2.00||20.00||300.00 |- | A5||182.27||1.65||A5||14.09||2.26||2.00||20.00||300.00 |- | A6||164.77||1.82||A6||13.92||2.26||2.00||20.00||300.00 |- | A7||112.93||2.66||A7||13.08||2.26||2.00||20.00||300.00 |- | A8||119.33||2.51||A8||13.23||2.26||2.00||20.00||300.00 |- | A9||188.90||1.59||A9||14.15||2.26||2.00||20.00||300.00 |- | A10||205.10||1.46||A10||14.28||2.26||2.00||20.00||300.00 |- | NTC||0.00||0.00||NTC||15.74||2.26||2.00||20.00||0.00 |- | #1||34.6||5.78||#1||9.96||2.26||2.00||20.00||200.00 |- | #2||30.4||6.58||#2||9.16||2.26||2.00||20.00||200.00 |- | #3||39.9||5.01||#3||10.73||2.26||2.00||20.00||200.00 |- | #4||41.4||4.83||#4||10.91||2.26||2.00||20.00||200.00 |- | #5||21.6||9.26||#5||6.48||2.26||2.00||20.00||200.00 |- | #6||30.9||6.47||#6||9.27||2.26||2.00||20.00||200.00 |- | #7||36.1||5.54||#7||10.20||2.26||2.00||20.00||200.00 |- | #8||34.4||5.81||#8||9.93||2.26||2.00||20.00||200.00 |- | NTC||0||0.00||NTC||15.74||2.26||2.00||20.00||0.00 |} ===== GP1V4 reaction mix ===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''22 rxn mix''' |- | 10X AmpLigase Buffer||2.00||44.00 |- | GP1_V4 (5.75ng/ul)||2.26||49.74 |- | Toal ||'''4.26'''|| |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h -> add 3ul SLN mix (1.5U/ul '''''Illumina's polymerase''''' (3U/reaction); 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 6h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. === SLN Mix === {| {{table}} | 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 (ul), total 48ul''' |- | Illumina's Polymerase||3||U/ul||1.5||U/ul||24.00 |- | AmpLigase||5||U/ul||0.5||U/ul||4.80 |- | dNTP||1||mM||100||uM||4.80 |- | 10x AmpLigase Buffer||10||x||1||x||4.80 |- | H2O||||||||||9.60 |} === Amplification === * Add 15% v/v captured DNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''22 rxn mix''' |- | Captured template||15.00||0.00 |- | 10uM AmpF6.4Sol||2.00||44.00 |- | 10uM AmpR6.3.IndX (X=1-16)||2.00||0.00 |- | 2X KAPA SYBR MM||50.00||1100.00 |- | H2O||31.00||682.00 |- | Total||100.00|| |} :- Aliquot 83, add 2ul of AmpR6.3.IndX, 15ul of template<br> :- Use the same PCR profile for library-free protocol :- Stopped reaction at 21 cycles {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Index''' |- | A1||Ind_41||A9||Ind_49||#1||Ind_57 |- | A2||Ind_42||A10||Ind_50||#2||Ind_58 |- | A3||Ind_43||NTC-1||Ind_51||#3||Ind_59 |- | A4||Ind_44||NTC-2||Ind_52||#4||Ind_60 |- | A5||Ind_45||NTC-PCR||Ind_53||#5||Ind_61 |- | A6||Ind_46||||||#6||Ind_62 |- | A7||Ind_47||||||#7||Ind_63 |- | A8||Ind_48||||||#8||Ind_64 |} * Verified PCR product in 6% TBE gel by loading 4ul of PCR product [[File:ZhangLab_2 2014-05-09 15hr 25min_ILMN_GP1V4_gel1_A1-A10_NTC.jpg| 400px]] <br> <br> [[File:ZhangLab_2 2014-05-09 15hr 27min_ILMN_GP1V4_gel2_no1-8_NTC.jpg| 400px]] * Perform AMPure bead purification (0.7X volume) * Elute with 55ul EB buffer * Verify and quantify DNA concentration in 6% TBE gel by loading 4ul of bead-purified DNA [[File:ZhangLab_2 2014-05-17 17hr 52min_PQ4ul_A1-A10_Exp5-07.jpg| 400px]]<br> <br> [[File:ZhangLab_2 2014-05-17 17hr 52min_PQ4ul_no1-no8_NTC_Exp5-07.jpg|400px]] == BSPP capture round2 using Stoffel Fragment == * I repeated experiment on samples A1-A10 using Stoffel fragment * Everything was the same except I increase amount of template to 400ng since I notice smaple A1-A10 had fainter intensity comparing to sample #1-#8 :- Use the same PCR profile for library-free protocol :- Stopped reaction at 22 cycles * Verified PCR product in 6% TBE gel by loading 4ul of PCR product [[File:ZhangLab_2 2014-05-16 18hr 43min_ILMN_GP1v4_A1-A8_4ulPCR.jpg| 400px]]<br> <br> [[File:ZhangLab_2 2014-05-16 18hr 43min_ILMN_GP1v4_A9-A10_4ulPCR.jpg| 120px]] * Perform AMPure bead purification (0.7X volume) * Elute with 55ul EB buffer * Verify and quantify DNA concentration in 6% TBE gel by loading 3ul of bead-purified DNA [[File:ZhangLab_2 2014-05-17 17hr 52min_PQ_3ul_A1-A10_Exp5-13.jpg|450px]]
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