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== Bisulfite conversion == * I did bisulfite converssion by using '''EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo''' and followed the protocol of the kit * I did two replicates for each amount ==== Bisulfite conversion set up ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | 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)''' |- | HAPMAP-A1||71.60||3.49||16.51||130.00||150.00||250.00 |- | HAPMAP-A1||71.60||6.98||13.02||130.00||150.00||500.00 |- | HAPMAP-B1||88.10||5.68||14.32||130.00||150.00||500.00 |- | HAPMAP-B1||88.10||8.51||11.49||130.00||150.00||750.00 |} ''' 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 2ul for ssDNA Qubit assay. The volume left after assay ~13ul. Next time I may use only 1ul for Qubit assay to save DNA sample for capture, since 1ul pipetting is accurate enough and the concentration is in the measurable range. ==== ssDNA Qubit assay result ==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Start amount (ng)''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yield in 15ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Yield left after <br>Qubit (13ul)''' | align="center" style="background:#f0f0f0;"|'''% Recovery in 15ul''' |- | 1) HAPMAP-A1||250||6.07||91.05||78.91||36.42 |- | 2) HAPMAP-A1||250||5.89||88.35||76.57||35.34 |- | 3) HAPMAP-A1||500||13.70||205.50||178.10||41.10 |- | 4) HAPMAP-A1||500||12.50||187.50||162.50||37.50 |- | 5) HAPMAP-B1||500||15.90||238.50||206.70||47.70 |- | 6) HAPMAP-B1||500||17.00||255.00||221.00||51.00 |- | 7) HAPMAP-B1||750||25.90||388.50||336.70||51.80 |- | 8) HAPMAP-B1 ||750||25.20||378.00||327.60||50.40 |- | BSPP_Blueprint||NA||25.40||1651.00||NA||NA |} * Note: I measure the BSPP_Blueprint probe using ssDNA Qubit assay as well. The concentration was about half of the concentration by PAGE quantification. This makes sense because the low mass ladder always showed a smear in TBU. This caused the actual amount in each band was lower than it's supposed to be and the quantification will be overestimated. I will use conc. from Qubit for probe concentration as well. ==== Discussion about bisulfite conversion ==== * Will be added === Experimental plan II === * I will do BSPP capture by varying bisulfite converted gDNA amount to see the minimum amount of 12K BSPP probe set that work well for capture experiment. * I will also vary the probe: target ratio. As I know that the DMR330k BSPP probe set works well with the ratio ~100:1 - ~200:1, but I have adjusted the probe:target ratio in each set. In addition the DMR330K are muck more larger than the BSPP_Blueprint probe set. I may need to increase the probe:target ratio. * I will set up BSPP capture with template amount 50ng, 100ng, 150ng and 300ng (200-300ng is the amount that I usually used and worked well for previous experiment) * Each template amount will be captured with 200:1 and 500:1 probe:target. I actually should have done 1000:1 for comparison too.
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