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==Bisulfite Conversion using Zymo EZ DNA MEthylation-Lightning Kit (07/15/2015)== * Concentration based on [[Noi/NOTES/2014-10-22]] [[Noi/NOTES/2014-11-30]] {| class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" |- style="font-size:12pt" align="center" valign="bottom" | width="43" height="15" | Sample | width="105" | Concentration ng/ul | width="78" | 1ng Volume ul | width="65" | 20ul H2O ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | NC2 | align="center" | 0.325 | align="center" | 3.08 | align="center" | 16.92 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | NC3 | align="center" | 0.538 | align="center" | 1.86 | align="center" | 18.14 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 6P1 | align="center" | 0.2 | align="center" | 5.00 | align="center" | 15.00 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 7P8 | align="center" | 0.167 | align="center" | 5.99 | align="center" | 14.01 |} * We do not have 1ng of DNA {| class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" |- style="font-size:12pt" align="center" valign="bottom" | width="43" height="15" | Sample | width="105" | Concentration ng/ul | width="78" | Volume ul | width="65" | 20ul H2O ul | width="89" | Amount Used ng |- style="font-size:12pt" align="center" valign="bottom" | height="15" | NC2 | align="center" | 0.325 | align="center" | 3.08 | align="center" | 16.92 | align="center" | 1 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | NC3 | align="center" | 0.538 | align="center" | 1.86 | align="center" | 18.14 | align="center" | 1 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 6P1 | align="center" | 0.2 | align="center" | 3.3 | align="center" | 16.7 | align="center" | 0.66 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 7P8 | align="center" | 0.167 | align="center" | 3.5 | align="center" | 16.50 | align="center" | 0.58 |} * Add 130ul lighting conversion reagent to 20ul of sample, compensate with water if less than 20ul. * Vortex and centrifuge. * (98C 8min > 54C 60min > 10C hold) * Add 600ul of M-Binding Buffer to column with collection tube. * Load the sample to column. Close the cap and mix by inverting the column several times. * Centrifuge at full speed for 30s. Discard flow through. * Add 100 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30s. * Add 200 μl of L-Desulphonation Buffer to the column and let stand at RT for 20 mins. After the incubation, centrifuge at full speed for 30 seconds. * Add 200 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30 seconds. * Repeat this wash step. * Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA. * The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. -80C for long term storage.
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