Matt:LabNotes/2017-4-22: Difference between revisions
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>Mzcai (Created page with "=In tube SplintR Test with formamide and ET SSB= ==Test Conditions== #Standard: SplintR only #SplintR incubated for 30min instead of 15min #SplintR + 10% formamide #SplintR +...") |
>Mzcai mNo edit summary |
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Line 11: | Line 11: | ||
#Positive Control: Ampligase | #Positive Control: Ampligase | ||
==Padlock Probes== | *For each test conditions have | ||
**one sample with ALL padlock probes and template | |||
***Should see amplification | |||
**one sample with all padlock probes and template MINUS ppMALAT1 | |||
***Should not see amplification | |||
==Padlock Probes and Template== | |||
*Same as [[Matt:LabNotes/2017-4-7]] with the addition of ppMALAT1 for consistency | *Same as [[Matt:LabNotes/2017-4-7]] with the addition of ppMALAT1 for consistency | ||
*ppCUX2 | *ppCUX2 | ||
Line 18: | Line 24: | ||
*ppGFAP | *ppGFAP | ||
*ppMALAT1 | *ppMALAT1 | ||
* | *Template for ppMALAT1: MALAT1_template | ||
==Protocol== | ==Protocol== | ||
#Add | #Combine padlock probes and template in 1X Ligase buffer and possibly formamide | ||
# | #Add mineral oil on top | ||
#Incubate at 55C for 17hr | |||
#To sample 7 (2-step ET SSB) add 200ng (0.4ul) of ET SSB and keep at 55C for 30min | |||
#To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min | |||
#*Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O | |||
#After sample 7 has ET SSB for 30min move samples 1-7 to 37C | |||
#Add 3ul SplintR Mix and incubate 15min | |||
#*SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O | |||
#*30min incubation for sample 2 | |||
#*Also add 0.4ul ET SSB to sample 6 | |||
#Put all samples on ice and add 2ul Exo I/III mix | |||
#*Also add 0.4ul ET SSB to samples 1-5 and 8 | |||
#Incubate at 37C for 1hr | |||
#qPCR all 16 samples | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''16X Volume''' | |||
|- | |||
| Captured template||5||0 | |||
|- | |||
| 10uM ISB_CA_AF||0.4||6.4 | |||
|- | |||
| 10uM ISB_CA_AR.T1||0.4||6.4 | |||
|- | |||
| 2X KAPA SYBG MM||12.5||200 | |||
|- | |||
| H2O||6.7||107.2 | |||
|- | |||
| Total||25||320 | |||
|} | |||
*Aliquot 20ul from 16X master mix and add 5ul captured template | |||
Program | |||
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min | |||
===Mistakes=== | |||
*Forgot to heat inactivate (incubate at 94C 10min) after ligation and before Exo I/III | |||
*Accidentally added 3ul 10X SplintR Buffer to samples 1-3 that HAVE ppMALAT1 | |||
**Still added 3ul SplintR Mix afterwards | |||
*Do zymo columns after Exo I/III next time |
Revision as of 23:11, 23 April 2017
In tube SplintR Test with formamide and ET SSB
Test Conditions
- Standard: SplintR only
- SplintR incubated for 30min instead of 15min
- SplintR + 10% formamide
- SplintR + 20% formamide
- SplintR + 30% formamide
- SplintR + 250ng ET SSB
- SplintR + 250ng ET SSB added first
- Positive Control: Ampligase
- For each test conditions have
- one sample with ALL padlock probes and template
- Should see amplification
- one sample with all padlock probes and template MINUS ppMALAT1
- Should not see amplification
- one sample with ALL padlock probes and template
Padlock Probes and Template
- Same as Matt:LabNotes/2017-4-7 with the addition of ppMALAT1 for consistency
- ppCUX2
- ppBCL11B
- ppRELN_1
- ppGFAP
- ppMALAT1
- Template for ppMALAT1: MALAT1_template
Protocol
- Combine padlock probes and template in 1X Ligase buffer and possibly formamide
- Add mineral oil on top
- Incubate at 55C for 17hr
- To sample 7 (2-step ET SSB) add 200ng (0.4ul) of ET SSB and keep at 55C for 30min
- To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min
- Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
- After sample 7 has ET SSB for 30min move samples 1-7 to 37C
- Add 3ul SplintR Mix and incubate 15min
- SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O
- 30min incubation for sample 2
- Also add 0.4ul ET SSB to sample 6
- Put all samples on ice and add 2ul Exo I/III mix
- Also add 0.4ul ET SSB to samples 1-5 and 8
- Incubate at 37C for 1hr
- qPCR all 16 samples
Components | 1X Volume | 16X Volume |
Captured template | 5 | 0 |
10uM ISB_CA_AF | 0.4 | 6.4 |
10uM ISB_CA_AR.T1 | 0.4 | 6.4 |
2X KAPA SYBG MM | 12.5 | 200 |
H2O | 6.7 | 107.2 |
Total | 25 | 320 |
- Aliquot 20ul from 16X master mix and add 5ul captured template
Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
Mistakes
- Forgot to heat inactivate (incubate at 94C 10min) after ligation and before Exo I/III
- Accidentally added 3ul 10X SplintR Buffer to samples 1-3 that HAVE ppMALAT1
- Still added 3ul SplintR Mix afterwards
- Do zymo columns after Exo I/III next time