EricChu:LabNotesMDA/2013-5-3

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MDA of genomic DNA in MDA Device v.7[edit]

  • 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.
  1. 2ul 2.5ng/ul mouse gDNA was added to 3ul ALS. Three minute incubation then kept on ice.
  2. prepare mastermix (22.5ul volume)
    1. 10x buffer: 2.33ul
    2. N6 primer: 8.45ul (<-1.5x normal)
    3. dNTP: 1.688ul (<-1.5x normal)
    4. 8%Tween20: 2.33ul
    5. Phi29 (high concentration): 0.84ul (<-3x normal)
    6. H20: 4.53ul
  3. split mastermix into 3 parts
    1. 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.
    2. negative control: Added 0.28ul H20 into 8ul mastermix. 30 degree C for 5 hours.
    3. remaining 5ul was used to load the MDA device
  4. load ALS+NS first, then NS. MDA mastermix was loaded last and filled all the chambers from top to bottom.

MDA Device V.7[edit]

File:MDAdeviceV7.jpg

qPCR using B2 primers[edit]

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