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=== August 22 === ==== Pipet shearing test by qMDA ==== Motivation: Several qMDA tests have failed to detect extracted amplicons above a background contamination level of ~1 fg. * We expect ~1 ng from overnight microwell MDA. * All ladders (10 ng, 1 ng, 100 pg, 10 pg) were treated the same as samples after extraction. All ladder amounts showed expected CT values. * We suspect that qMDA is inefficient on small templates (hyperbranch structure forms inefficiently), and that the glass pipets are shearing the sample. Objective: Test whether shearing the template through glass pipets inhibits qMDA. We have three conditions: * No shear (ladder). * Shear 1X (pipet up and down), 2 bio replicates. * Shear 5X, 2 bio replicates. Denaturation and neutralization in 2X volume, split into white strip tubes and add master mix. * 8 samples, in duplicate (16 total): ** 10 ng ** 1 ng ** 100 pg ** 10 pg ** 1 ng, 1X shear, rep 1 ** 1 ng, 1X shear, rep 2 ** 1 ng, 5X shear, rep 1 ** 1 ng, 5X shear, rep 2 Protocol # 2 ul sample plus 3 ul ALS, 3 min @ RT. # On ice, add 3 ul NS --> 8 ul. #Split, 4 ul and 4 ul in white tubes. # Add 16 ul master mix. * Master mix (18X): ** 36 ul Phi29 buffer. ** 18 ul thio N6. ** 14.4 ul RepliPhi dNTP. ** 2.88 50X SYBR. ** 18 ul Phi29. ** 198.72 ul H2O. 30 degC, measure every 6 min, up to 99 cycles (or completion). Conclusion: No difference between 1 ng ladder, 1 ng 1X shear, and 1 ng 5X shear. Pipet shearing does not appear to affect efficiency of qMDA. [[File: 2013_08_22 qmda shear test.xls|2013_08_22 qmda shear test]]
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