EricChu:LabNotesMDA/2013-5-3: Difference between revisions

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*note: Since no mouse amplicon was detected by qPCR in single cell experiment, mouse genomic DNA was used to verify the device.
*note: Since no mouse amplicon was detected by qPCR in single cell experiment, mouse genomic DNA was used to verify the device.


Protocol
Protocol adjusted to the protocol in Apr2
# 2ul 2.5ng/ul mouse gDNA was added to 3ul ALS. Three minute incubation then kept on ice.
# 2ul 2.5ng/ul mouse gDNA was added to 3ul ALS. Three minute incubation then kept on ice.
# prepare mastermix (22.5ul volume)
# prepare mastermix (22.5ul volume)
Line 28: Line 28:
[[File:05-03-2013samples.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)
* 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
[[File:05-03-2013qPCR.jpg]]
[[File:05-03-2013qPCR.jpg]]
[[File:Apr2to5qPCRVerticalMDA.jpg]]

Revision as of 22:05, 3 May 2013

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

  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

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.

File:05-03-2013std+samples.jpg

  • Both positive control and negative control were no amplified.

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

File:05-03-2013qPCR.jpg File:Apr2to5qPCRVerticalMDA.jpg