EricChu:LabNotesMDA/2013-5-14: Difference between revisions

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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.
[[File:05142013.jpg]]
[[File:05142013.jpg]]


[[File:05142013TotalDNA.jpg]]
[[File:05142013TotalDNA.jpg]]

Revision as of 21:43, 15 May 2013

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.
  1. 1ul 2.5ng/ul mouse gDNA was added to 1ul ALS. Added 1ul NS after Three minute incubation, then kept on ice. [Genomic DNA]
  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 0.46ul of Genomic DNA to 13ul mastermix. Transferred 8ul to another PCR tube. 30 degree C for 15 hours.
    2. remaining volume was used to load the MDA device
    3. negative control: Added 0.28ul H20 to 8ul mastermix. 30 degree C for 15 hours.

File:05142013.jpg

File:05142013TotalDNA.jpg