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.
- 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.