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==== Procedures ==== * Followed protocol of DNeasy Blood and tissue kit: [[http://mvz.berkeley.edu/egl/inserts/DNeasy_Blood_&_Tissue_Handbook.pdf]]. Page 28. * I added RNase A to remove RNA contamination :- Rui suggested that if the tissue is large, I should increase volume of ATL buffer ~3X. :- I added 180*3ul = 540ul of ATL buffer + 50ul of 10mg/mL RNase A (actually should right before Buffer AL) + 60ul of ProteinaseK :- Incubate at 56C for ~1h with shaking 900rpm/min and occasionally vigorously vortexing for every 10min. :: I did not use homogenizer to homogenize tissue as it might be difficult to handle and it will generate a lot of bubbles. :- Anyway, Vortexing and shaking on thermo mixer is good enough to lyse the tissue. :- After the tissue is completely lysed, I split it into two tube (~330ul each) + added 330ul of Buffer AL * Eluted 1st time with 200ul AE Buffer * Eluted 2nd time with 50ul AE Buffer (total each column ~250ul * Pooled two tubes together --> total volume left ~480ul * Measure concentration with Qubit dsDNA HS assay using 2ul of extracted DNA in 200ul reaction (100x dilution). Samples PC-T_1 has very high concentration. I repeated measurement and reduced volume to 1ul.
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