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 | ||
* 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. | # 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 24: | Line 25: | ||
[[File:05-03-2013std.jpg]] | [[File:05-03-2013std.jpg]] | ||
* The five set of amplified curves were 1ng, 100pg, 10pg, 1pg, and NTC. | * 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:05-03-2013std+samples.jpg]] | ||
[[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. | |||
* I didn't get more products in bigger volume (May3). Is it about optimization? | |||
[[File:05-03-2013qPCR2.jpg]] | |||
[[File:Apr2to5qPCRVerticalMDA.jpg]] |
Latest revision as of 22:22, 3 May 2013
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.
- 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[edit]
qPCR using B2 primers[edit]
- 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?