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 m (→Protocol) |
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(5 intermediate revisions by the same user not shown) | |||
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#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 17: | Line 23: | ||
*ppRELN_1 | *ppRELN_1 | ||
*ppGFAP | *ppGFAP | ||
*ppMALAT1 | *ppMALAT1 | ||
** | **/5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT | ||
*Template for ppMALAT1: MALAT1_template | |||
**/5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT | |||
==Protocol== | ==Protocol== | ||
#Add | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
# | |- style="font-size:11pt" valign="bottom" | ||
| width="51" height="14" | Sample # | |||
| width="51" | Condition | |||
| width="195" | 100nM template + PP mix | |||
| width="140" | 55C 17hr | |||
| width="51" | 55C | |||
| width="51" | 37C | |||
| width="51" | Exo I/III | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 1 | |||
| SplintR 15min | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul SplintR Buffer + H2O to 30ul | |||
| - | |||
| Add 3ul SplintR Mix | |||
| 1ul Exo I + 1ul Exo II + 0.4ul ET SSB | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 2 | |||
| SplintR 30min | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul SplintR Buffer + H2O to 30ul | |||
| - | |||
| Add 3ul SplintR Mix | |||
| 1ul Exo I + 1ul Exo II + 0.4ul ET SSB | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 3 | |||
| SplintR + 10% formamide | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul SplintR Buffer + 3ul formamide + H2O to 30ul | |||
| - | |||
| Add 3ul SplintR Mix | |||
| 1ul Exo I + 1ul Exo II + 0.4ul ET SSB | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 4 | |||
| SplintR + 20% formamide | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul SplintR Buffer + 6ul formamide + H2O to 30ul | |||
| - | |||
| Add 3ul SplintR Mix | |||
| 1ul Exo I + 1ul Exo II + 0.4ul ET SSB | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 5 | |||
| SplintR + 30% formamide | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul SplintR Buffer + 9ul formamide + H2O to 30ul | |||
| - | |||
| Add 3ul SplintR Mix | |||
| 1ul Exo I + 1ul Exo II + 0.4ul ET SSB | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 6 | |||
| SplintR + ET SSB | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul SplintR Buffer + H2O to 30ul | |||
| - | |||
| Add 3ul SplintR Mix + 0.4ul ET SSB | |||
| 1ul Exo I + 1ul Exo II | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 7 | |||
| SplintR + 2step ET SSB | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul SplintR Buffer + H2O to 30ul | |||
| Add 0.4ul ET SSB | |||
| Add 3ul SplintR Mix | |||
| 1ul Exo I + 1ul Exo II | |||
|- style="font-size:11pt" valign="bottom" | |||
| align="right" height="14" | 8 | |||
| Ampligase | |||
| 1.5 or 1.8ul (0.3ul of each 10uM oligo) | |||
| 3ul Ampligase Buffer + H2O to 30ul | |||
| Add 3ul Ampligase Mix | |||
| - | |||
| 1ul Exo I + 1ul Exo II + 0.4ul ET SSB | |||
|} | |||
#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 | |||
==Results== | |||
[[Media:20170423_qPCR_PPcapture_SplintRformamideETSSB.xlsx|raw data here]]<br> | |||
[[File:20170423_qPCR_ppCapture_SplintRformamideETSSB.PNG|650px]] | |||
*Possible options: 10% formamide | |||
[[File:20170423_qPCR_PPcapture_SplintRformamideETSSB_ScatterPlot.PNG|450px]] | |||
*No difference between 15min and 30min SplintR incubation times | |||
*ET SSB added does increase the number of captured padlock probes | |||
**Adding ET SSB before SplintR vs during SplintR makes no difference | |||
*Next time do a ET SSB + 10% formamide sample | |||
*Next time try less template concentration... see if it can distinguish the smaller amount |
Latest revision as of 19:54, 19 May 2017
In tube SplintR Test with formamide and ET SSB[edit]
Test Conditions[edit]
- 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[edit]
- Same as Matt:LabNotes/2017-4-7 with the addition of ppMALAT1 for consistency
- ppCUX2
- ppBCL11B
- ppRELN_1
- ppGFAP
- ppMALAT1
- /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT
- Template for ppMALAT1: MALAT1_template
- /5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT
Protocol[edit]
Sample # | Condition | 100nM template + PP mix | 55C 17hr | 55C | 37C | Exo I/III |
1 | SplintR 15min | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul SplintR Buffer + H2O to 30ul | - | Add 3ul SplintR Mix | 1ul Exo I + 1ul Exo II + 0.4ul ET SSB |
2 | SplintR 30min | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul SplintR Buffer + H2O to 30ul | - | Add 3ul SplintR Mix | 1ul Exo I + 1ul Exo II + 0.4ul ET SSB |
3 | SplintR + 10% formamide | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul SplintR Buffer + 3ul formamide + H2O to 30ul | - | Add 3ul SplintR Mix | 1ul Exo I + 1ul Exo II + 0.4ul ET SSB |
4 | SplintR + 20% formamide | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul SplintR Buffer + 6ul formamide + H2O to 30ul | - | Add 3ul SplintR Mix | 1ul Exo I + 1ul Exo II + 0.4ul ET SSB |
5 | SplintR + 30% formamide | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul SplintR Buffer + 9ul formamide + H2O to 30ul | - | Add 3ul SplintR Mix | 1ul Exo I + 1ul Exo II + 0.4ul ET SSB |
6 | SplintR + ET SSB | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul SplintR Buffer + H2O to 30ul | - | Add 3ul SplintR Mix + 0.4ul ET SSB | 1ul Exo I + 1ul Exo II |
7 | SplintR + 2step ET SSB | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul SplintR Buffer + H2O to 30ul | Add 0.4ul ET SSB | Add 3ul SplintR Mix | 1ul Exo I + 1ul Exo II |
8 | Ampligase | 1.5 or 1.8ul (0.3ul of each 10uM oligo) | 3ul Ampligase Buffer + H2O to 30ul | Add 3ul Ampligase Mix | - | 1ul Exo I + 1ul Exo II + 0.4ul ET SSB |
- 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[edit]
- 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
Results[edit]
raw data here
File:20170423 qPCR ppCapture SplintRformamideETSSB.PNG
- Possible options: 10% formamide
File:20170423 qPCR PPcapture SplintRformamideETSSB ScatterPlot.PNG
- No difference between 15min and 30min SplintR incubation times
- ET SSB added does increase the number of captured padlock probes
- Adding ET SSB before SplintR vs during SplintR makes no difference
- Next time do a ET SSB + 10% formamide sample
- Next time try less template concentration... see if it can distinguish the smaller amount