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==MDA of genomic DNA in MDA Device v.7== *note: Since no mouse amplicon was detected by qPCR in single cell experiment, mouse genomic DNA was used to verify the device. Protocol adjusted to the protocol in Apr2 * With the new volume (23nl), the maximum amount of DNA possible is 9.26ng. # 2ul 2.5ng/ul mouse gDNA was added to 3ul ALS. Three minute incubation then kept on ice. # prepare mastermix (22.5ul volume) ## 10x buffer: 2.33ul ## N6 primer: 8.45ul (<-1.5x normal) ## dNTP: 1.688ul (<-1.5x normal) ## 8%Tween20: 2.33ul ## Phi29 (high concentration): 0.84ul (<-3x normal) ## H20: 4.53ul # split mastermix into 3 parts ## positive control: Added 1ul ALS+DNA from step one to 0.6ul NS. Then transferred 0.28ul of the mix to 8ul mastermix. 30 degree C for 5 hours. ## negative control: Added 0.28ul H20 into 8ul mastermix. 30 degree C for 5 hours. ## remaining 5ul was used to load the MDA device # load ALS+NS first, then NS. MDA mastermix was loaded last and filled all the chambers from top to bottom. ==MDA Device V.7== [[File:MDAdeviceV7.jpg]] ==qPCR using B2 primers== [[File:05-03-2013std.jpg]] * The five set of amplified curves were 1ng, 100pg, 10pg, 1pg, and NTC. * Both positive control and negative control were not amplified. * Why didn't the positive control show up? I have seen this before; if I dilute the positive control 100-1000x, it would be detected. What is inhibiting the qPCR reaction? [[File:05-03-2013std+samples.jpg]] [[File:05-03-2013samples.jpg]] * All samples fell in between 10pg and 100pg. While 1ul was used for qPCR, the total DNA per each sample was multiplied by 5. (5ul samples were collected) * Result from qPCR and comparison to Apr2 result. * I didn't get more products in bigger volume (May3). Is it about optimization? [[File:05-03-2013qPCR2.jpg]] [[File:Apr2to5qPCRVerticalMDA.jpg]]
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