Matt:LabNotes/2014-1-15: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (Created page with "===Exo I/III in situ to check 2ndary Rolony Ampligase reaction=== *Currently 2nd Rolony fabrication is inefficient or unsuccessful **Need a positive and negative control (curr...") |
>Mzcai m (→Procedure) |
||
Line 10: | Line 10: | ||
====Procedure==== | ====Procedure==== | ||
*Use 6 wells with 1st Rolonies from 96-well plate: | *Use 6 wells with 1st Rolonies from 96-well plate: | ||
*2 Positive control: +Ampligase, -Exo (expect to see lots of signal like previous experiments) | *2 Positive control (C7 & D7): +Ampligase, -Exo (expect to see lots of signal like previous experiments) | ||
*2 Negative control: -Ampligase, +Exo (expect to see no signal) | *2 Negative control (E7 & F7): -Ampligase, +Exo (expect to see no signal) | ||
*2 Experimental: +Ampligase, +Exo (expect to see a % of a positive control, hopefully close to 100%) | *2 Experimental (D9 & E9): +Ampligase, +Exo (expect to see a % of a positive control, hopefully close to 100%) | ||
'''Determine Number of ppMALAT1 targets''' | '''Determine Number of ppMALAT1 targets''' | ||
#Hybridize | #Hybridize 2nM ppMALAT1 (in 2X SSC) | ||
##Preheat to 70C for 5min and then add 50ul to each well | ##Preheat to 70C for 5min and then add 50ul to each well | ||
##Incubate 50C for 10min | ##Incubate 50C for 10min | ||
##Wash once with 2X SSC | ##Wash once with 2X SSC | ||
#Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC | #Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul) | ||
##Preheat to 60C for 5min and then add 50ul to each well | ##Preheat to 60C for 5min and then add 50ul to each well | ||
##Incubate RT for 15min | ##Incubate RT for 15min | ||
##Wash 2 times with 2X SSC | ##Wash 2 times with 2X SSC | ||
#Image (ATTO488) | #Image (ATTO488) | ||
#Strip with 80% formamide for | #Strip with 80% formamide for 10min @50C | ||
'''Ampligase Experiment''' | '''Ampligase Experiment''' | ||
#Hybridize | #Hybridize 2nM ppMALAT1 (in 2X SSC) | ||
##Preheat to 70C for 5min and then add 50ul to each well | ##Preheat to 70C for 5min and then add 50ul to each well | ||
##Incubate 50C for 10min | ##Incubate 50C for 10min | ||
Line 35: | Line 35: | ||
##Incubate 50C for 4hrs | ##Incubate 50C for 4hrs | ||
#Wash twice with 1X PBS | #Wash twice with 1X PBS | ||
#Add 2.5ul Exo I and 2.5ul Exo III | #Add 50ul Exo I/III to each well (2.5ul 20U/ul Exo I and 2.5ul 200U/ul Exo III) | ||
##Positive control: | ##Exo I/III mix: 20ul 10X Exo III Buffer + 10ul Exo I + 10ul Exo III + 160ul H2O | ||
##Positive control: 50ul of (10ul 10X Exo III Buffer + 90ul H2O) | |||
##Incubate 37C for 2hrs | ##Incubate 37C for 2hrs | ||
#Wash twice with 1X PBS preheated to 94C | #Wash twice with 1X PBS preheated to 94C | ||
##Let sit for 1min before aspirating | ##Let sit for 1min before aspirating | ||
#Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC | #Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul) | ||
##Preheat to 60C for 5min and then add 50ul to each well | ##Preheat to 60C for 5min and then add 50ul to each well | ||
##Incubate RT for 15min | ##Incubate RT for 15min | ||
##Wash 2 times with 2X SSC | ##Wash 2 times with 2X SSC | ||
#Image (ATTO488) | #Image (ATTO488) |
Latest revision as of 23:09, 17 January 2014
Exo I/III in situ to check 2ndary Rolony Ampligase reaction[edit]
- Currently 2nd Rolony fabrication is inefficient or unsuccessful
- Need a positive and negative control (currently working on making positive control by pre-circularizing ppMALAT1)
- Trying to determine whether Ampligase reaction or 2nd RCA is the limiting step
- How to separate Ampligase step from 2nd RCA step?
- Add Exo I/III to cells after Ampligase step to digest non-circular DNA
- Then can use Shifted FISSEQ_2ndRCAprimerDye to detect the padlock probes that are left (and therefore circularized)
- By using positive and negative control, can determine yield of Ampligase step
Procedure[edit]
- Use 6 wells with 1st Rolonies from 96-well plate:
- 2 Positive control (C7 & D7): +Ampligase, -Exo (expect to see lots of signal like previous experiments)
- 2 Negative control (E7 & F7): -Ampligase, +Exo (expect to see no signal)
- 2 Experimental (D9 & E9): +Ampligase, +Exo (expect to see a % of a positive control, hopefully close to 100%)
Determine Number of ppMALAT1 targets
- Hybridize 2nM ppMALAT1 (in 2X SSC)
- Preheat to 70C for 5min and then add 50ul to each well
- Incubate 50C for 10min
- Wash once with 2X SSC
- Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul)
- Preheat to 60C for 5min and then add 50ul to each well
- Incubate RT for 15min
- Wash 2 times with 2X SSC
- Image (ATTO488)
- Strip with 80% formamide for 10min @50C
Ampligase Experiment
- Hybridize 2nM ppMALAT1 (in 2X SSC)
- Preheat to 70C for 5min and then add 50ul to each well
- Incubate 50C for 10min
- Wash once with 2X SSC
- Add 0.05U/ul Ampligase to Positive and Exprimental (2ul Ampligase + 20ul 10X Buffer + 178ul H2O)
- Negative control: (90ul H2O + 10ul 10X buffer)
- Incubate 50C for 4hrs
- Wash twice with 1X PBS
- Add 50ul Exo I/III to each well (2.5ul 20U/ul Exo I and 2.5ul 200U/ul Exo III)
- Exo I/III mix: 20ul 10X Exo III Buffer + 10ul Exo I + 10ul Exo III + 160ul H2O
- Positive control: 50ul of (10ul 10X Exo III Buffer + 90ul H2O)
- Incubate 37C for 2hrs
- Wash twice with 1X PBS preheated to 94C
- Let sit for 1min before aspirating
- Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul)
- Preheat to 60C for 5min and then add 50ul to each well
- Incubate RT for 15min
- Wash 2 times with 2X SSC
- Image (ATTO488)