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== Bisulfite conversion == I quantified gDNA concentration by Qubit dsDNA assay. Note that the volume of each sample they sent us was variied so I can not summarize total amount of each sample. However, based on concentration by Qubit the total amount we got of each sample less than 1ug as they claimed. Therefore I may not have enough gDNA for the second trial even I used only 500ng for bisulfite conversion. Also, there were few samples which total amount is very low so total amount for bis-cvt less than 300ng. * I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''WellIDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' |- | 1||A1||37.71||13.26||6.74||130.00||150.00||500.00 |- | 2||B1||39.43||12.68||7.32||130.00||150.00||500.00 |- | 3||C1||50.57||9.89||10.11||130.00||150.00||500.00 |- | 4||D1||34.86||14.34||5.66||130.00||150.00||500.00 |- | 5||E1||56.00||8.93||11.07||130.00||150.00||500.00 |- | 6||F1||44.29||11.29||8.71||130.00||150.00||500.00 |- | 7||G1||73.71||6.78||13.22||130.00||150.00||500.00 |- | 8||H1||29.71||16.83||3.17||130.00||150.00||500.00 |- | 9||A2||40.29||12.41||7.59||130.00||150.00||500.00 |- | 10||B2||33.43||14.96||5.04||130.00||150.00||500.00 |- | 11||C2||44.86||11.15||8.85||130.00||150.00||500.00 |- | 12||D2||52.86||9.46||10.54||130.00||150.00||500.00 |- | **13||E2||11.34||9.00||11.00||130.00||150.00||102.09 |- | *14||F2||36.86||9.00||11.00||130.00||150.00||331.71 |- | **15||G2||26.74||9.00||11.00||130.00||150.00||240.69 |- | *16||H2||50.00||9.00||11.00||130.00||150.00||450.00 |- | 17||A3||34.00||14.71||5.29||130.00||150.00||500.00 |- | 18||B3||31.14||16.06||3.94||130.00||150.00||500.00 |- | 19||C3||26.46||18.90||1.10||130.00||150.00||500.00 |- | *20||D3||20.29||20.00||0.00||130.00||150.00||405.71 |- | *21||E3||18.23||20.00||0.00||130.00||150.00||364.57 |- | 22||F3||29.43||16.99||3.01||130.00||150.00||500.00 |- | 23||G3||47.71||10.48||9.52||130.00||150.00||500.00 |- | 24||H3||32.57||15.35||4.65||130.00||150.00||500.00 |- | 25||A4||29.14||17.16||2.84||130.00||150.00||500.00 |- | *26||B4||19.60||20.00||0.00||130.00||150.00||392.00 |- | 27||C4||24.31||20.00||0.00||130.00||150.00||486.29 |- | 28||D4||23.66||20.00||0.00||130.00||150.00||473.14 |- | 29||E4||47.43||10.54||9.46||130.00||150.00||500.00 |- | 30||F4||76.86||6.51||13.49||130.00||150.00||500.00 |- | 31||G4||40.00||12.50||7.50||130.00||150.00||500.00 |- | 32||H4||32.29||15.49||4.51||130.00||150.00||500.00 |- | 33||A5 (12878)||58.00||8.62||11.38||130.00||150.00||500.00 |- | 34||B5 (12878)||59.00||8.47||11.53||131.00||151.00||500.00 |} ''' Program '''<br> 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold * Eluted bisulfite converted DNA form the bead with 20ul H2O * I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul. ==== ssDNA Qubit assay result ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''WellIDs''' | align="center" style="background:#f0f0f0;"|'''Start amount (ng)''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yield in 17ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Yield left after<br>Qubit (15ul)''' | align="center" style="background:#f0f0f0;"|'''% Recovery in 17ul''' |- | 1||A1||500.00||21.00||357.00||315.00||71.40 |- | 2||B1||500.00||24.40||414.80||366.00||82.96 |- | 3||C1||500.00||19.10||324.70||286.50||64.94 |- | 4||D1||500.00||22.60||384.20||339.00||76.84 |- | 5||E1||500.00||20.60||350.20||309.00||70.04 |- | 6||F1||500.00||21.20||360.40||318.00||72.08 |- | 7||G1||500.00||22.00||374.00||330.00||74.80 |- | 8||H1||500.00||22.20||377.40||333.00||75.48 |- | 9||A2||500.00||20.60||350.20||309.00||70.04 |- | 10||B2||500.00||24.00||408.00||360.00||81.60 |- | 11||C2||500.00||19.06||324.02||285.90||64.80 |- | 12||D2||500.00||18.78||319.26||281.70||63.85 |- | 13||E2||102.09||2.06||35.02||30.90||34.30 |- | 14||F2||331.71||8.08||137.36||121.20||41.41 |- | 15||G2||240.69||6.50||110.50||97.50||45.91 |- | 16||H2||450.00||12.32||209.44||184.80||46.54 |- | 17||A3||500.00||11.56||196.52||173.40||39.30 |- | 18||B3||500.00||12.24||208.08||183.60||41.62 |- | 19||C3||500.00||11.44||194.48||171.60||38.90 |- | 20||D3||405.71||7.92||134.64||118.80||33.19 |- | 21||E3||364.57||8.10||137.70||121.50||37.77 |- | 22||F3||500.00||11.38||193.46||170.70||38.69 |- | 23||G3||500.00||13.96||237.32||209.40||47.46 |- | 24||H3||500.00||19.72||335.24||295.80||67.05 |- | 25||A4||500.00||21.20||360.40||318.00||72.08 |- | 26||B4||392.00||23.00||391.00||345.00||99.74 |- | 27||C4||486.29||23.80||404.60||357.00||83.20 |- | 28||D4||473.14||31.40||533.80||471.00||112.82 |- | 29||E4||500.00||20.80||353.60||312.00||70.72 |- | 30||F4||500.00||10.46||177.82||156.90||35.56 |- | 31||G4||500.00||47.00||799.00||705.00||159.80 |- | 32||H4||500.00||56.00||952.00||840.00||190.40 |- | 33||A5 (12878)||500.00||24.00||408.00||360.00||81.60 |- | 34||B5 (12878)||500.00||24.60||418.20||369.00||83.64 |} * There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting. * Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.
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