EricChu:LabNotesMDA/2013-5-3: Difference between revisions
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## remaining 5ul was used to load the MDA device | ## 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. | # load ALS+NS first, then NS. MDA mastermix was loaded last and filled all the chambers from top to bottom. | ||
[[File:MDAdeviceV7.jpg | |||
==MDA Device V.7== | |||
[[File:MDAdeviceV7.jpg]] | |||
==qPCR using B2 primers== | |||
[[File:05-03-2013std.jpg]] | [[File:05-03-2013std.jpg]] | ||
* The five set of amplified curves were 1ng, 100pg, 10pg, 1pg, and NTC. | |||
[[File:05-03-2013std+samples.jpg]] | [[File:05-03-2013std+samples.jpg]] | ||
* Both positive control and negative control were no amplified. | |||
[[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) |
Revision as of 21:53, 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
- 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
qPCR using B2 primers
- 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.
- 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)