EricChu:LabNotesMDA/2013-3-28
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Estimate Maximum Amplificaiton in the Whole Vertical MDA Device[edit]
- I've planned to convert all downstream SYBR gold visualizing chambers and mixing chambers to MDA reaction chambers.
- The total volume of MDA rxn increased from 1.7nl to 8.65nl
File:Vertical Test Device No Ring.jpg
- Amount of dNTPs = 1.25mM x 8.26nl = 6.22E12 nucleotides = 3.40ng maximum
- The poor result behind the previous MEF cell experiment was probably due to poor pH condition, raising from the mismatch of ALS and NS volume. Increasing the mastermix to denatured DNA ratio seemed to give better MDA reaction.
- The following images were taken from the 2nd MEF cell experiment.
- Chamber 1 and 2 initially had very little DNA because the whole volume was pushed to the far end (Chamber 24). Chamber 23 and 24 were completely filled with ALS+NS+DNA at the beginning. Amplification looked better in chambers with initially less ALS+NS+DNA (chamber 1 and 2). It might due to the increased amount of mastermix, which diluted the ALS+NS+DNA, to reach a more neutral pH for MDA reaction.
- In 3/22/2013 images, the DNA clusters were individual and scattered everywhere. Genomic DNA was initially in fragments and clusters were everywhere.
- In today's images, single mouse cell amplification showed hints of long stains of DNA for those clusters were linked together.
- 2-hour and 5-hour images showed that the amplification was complete in 2 hours.