EricChu:LabNotesMDA/2013-5-14
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MEF Genomic DNA Amplification in MDA Device v.7
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.