Hosuk:LabNotes/2014-1-19: Difference between revisions
Jump to navigation
Jump to search
>Hosuki78 No edit summary |
>Hosuki78 No edit summary |
||
(One intermediate revision by the same user not shown) | |||
Line 14: | Line 14: | ||
====Procedure==== | ====Procedure==== | ||
*Experiment Plan | *Experiment Plan | ||
*#Anneal Padlock probes – | *#Anneal Padlock probes – 4 probes | ||
*#Add SNL mix (Ampligase + | *#*EGFR T790M(Wild, Mutant), EGFR L858R(Wild, Mutant) | ||
*#Incubate 50C for | *#*50nM each in 2x SSC + 30% Formamide (2uL each) | ||
*#*Preheat probes at 80C for 5min, incubate at 50C for 4hr. | |||
*#Add SNL mix (Ampligase + KlenTaq enzyme mix) | |||
*#Incubate 50C for 20hr. | |||
*#Anneal Primers for 2nd RCA | *#Anneal Primers for 2nd RCA | ||
*#Run RCA reaction for overnight( | *#Run RCA reaction for overnight(20hr.) | ||
*#Anneal dcProbes, and Image | *#Anneal dcProbes, and Image | ||
[[File:ReactionMix_Ampligase_KlenTaq.png| | |||
[[File:ReactionMix_Ampligase_KlenTaq.png|600px]] |
Latest revision as of 00:38, 20 January 2014
NCIH1975 : Mutation Detection[edit]
Padlock Probes[edit]
- Padlock probes selection
- 1)EGFR T790M
- Wild : detected by dcProbe1
- Mutant : detected by dcProbe3 or dcProbe4
- 2)EGFR L858R
- Wild : detected by dcProbe4
- Mutant : detected by dcProbe2
Procedure[edit]
- Experiment Plan
- Anneal Padlock probes – 4 probes
- EGFR T790M(Wild, Mutant), EGFR L858R(Wild, Mutant)
- 50nM each in 2x SSC + 30% Formamide (2uL each)
- Preheat probes at 80C for 5min, incubate at 50C for 4hr.
- Add SNL mix (Ampligase + KlenTaq enzyme mix)
- Incubate 50C for 20hr.
- Anneal Primers for 2nd RCA
- Run RCA reaction for overnight(20hr.)
- Anneal dcProbes, and Image
- Anneal Padlock probes – 4 probes