EricChu:LabNotesMDA/2013-5-14
Jump to navigation
Jump to search
MEF Genomic DNA Amplification in MDA Device v.7 (incubated with N6 and BSA)[edit]
The MDA chambers were incubated with the mix of 1ul 200uM N6 and 4ul 0.1% BSA for 15min. It took about 2 hours to dry the channels with air. The device was then UVed for 15mins prior to load the MDA reagents.
Protocol adjusted to the protocol in Apr2
- With the new volume (23nl), the maximum amount (based on dNTP concentration) of DNA possible is 9.26ng.
- 1ul 2.5ng/ul mouse gDNA was added to 1ul ALS. Added 1ul NS after Three minute incubation, then kept on ice. [Genomic DNA]
- 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 0.46ul of Genomic DNA to 13ul mastermix. Transferred 8ul to another PCR tube. 30 degree C for 15 hours.
- remaining volume was used to load the MDA device
- negative control: Added 0.28ul H20 to 8ul mastermix. 30 degree C for 15 hours.
First 13 samples were collected in about 6ul volume each. 1ul of each sample was used for qPCR of B2 primers. The total amount per chamber was ranging from 1.02ng to 2.64ng. This is the first time I've seen so much DNA product.
Previous Result of the Same Recipe (Without N6 and BSA incubation)[edit]
This is a reminder of the result on May 3rd. ALS+DNA was mixed with NS in the device. The collect samples were in the range of 10pg to 100pg