Matt:LabNotes/2017-5-8: Difference between revisions
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#Combine padlock probes and template in 1X Ligase buffer and possibly formamide | #Combine padlock probes and template in 1X Ligase buffer and possibly formamide | ||
#Add mineral oil on top | #Add mineral oil on top | ||
#Incubate at 55C for | #Incubate at 55C for 18hr | ||
#To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min | #To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min | ||
#*Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O | #*Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O | ||
# | #Move samples 1-7 to 37C | ||
#Add 3ul SplintR Mix and incubate 15min | #Add 3ul SplintR Mix and incubate 15min | ||
#*SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O | #*SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O | ||
#*Also add 0.4ul ET SSB to sample 7 | |||
#*Also add 0.4ul ET SSB to sample | #Incubate at 94C for 10min | ||
#Put all samples on ice and add 2ul Exo I/III mix | #Put all samples on ice and add 2ul Exo I/III mix | ||
#*Also add 0.4ul ET SSB to samples 1- | #*Also add 0.4ul ET SSB to samples 1-6 and 8 | ||
#Incubate at 37C for 1hr | #Incubate at 37C for 1hr | ||
#Incubate at 94C for 10min | |||
#qPCR all 16 samples | #qPCR all 16 samples | ||
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| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' |
Revision as of 20:39, 9 May 2017
Second Try: in tube SplintR Test with formamide and ET SSB
- First try showed SplintR with 10% formamide had the best sensitivity and specificity of the conditions tried
- Will try varying formamide concentration around 10%
- Will qPCR with lower starting conc to get more accurate measurement
Test Conditions
- Standard: SplintR only
- SplintR + 5% formamide
- SplintR + 7.5% formamide
- SplintR + 10% formamide
- SplintR + 12.5% formamide
- SplintR + 15% formamide
- SplintR + 10% formamide + 250ng ET SSB
- 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 with NO MALAT1 template
- Should not see amplification
- one sample with ALL padlock probes and template
Padlock Probes and Template
- ppCUX2
- ppBCL11B
- ppRELN_1
- ppGFAP
- ppMALAT1
- /5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT
- Template for ppMALAT1: MALAT1_template
- /5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT
Protocol
Sample # | Condition | 10nM PP + Template | 10X Buffer | Formamide | H2O | Total |
1 | SplintR | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 0 | 25.5 or 25.2 | 30 |
2 | SplintR + 5% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 1.5 | 24 or 23.7 | 30 |
3 | SplintR + 7.5% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 2.25 | 23.25 or 22.95 | 30 |
4 | SplintR + 10% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 3 | 22.5 or 22.2 | 30 |
5 | SplintR + 12.5% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 3.75 | 21.75 or 21.45 | 30 |
6 | SplintR + 15% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 4.5 | 21 or 20.7 | 30 |
7 | SplintR + 10% formamide + ET SSB | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 3 | 22.5 or 22.2 | 30 |
8 | Ampligase | 1.5 or 1.8 (0.3ul of each 1uM oligo) | 3 | 0 | 25.5 or 25.2 | 30 |
- Combine padlock probes and template in 1X Ligase buffer and possibly formamide
- Add mineral oil on top
- Incubate at 55C for 18hr
- To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min
- Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
- 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
- Also add 0.4ul ET SSB to sample 7
- Incubate at 94C for 10min
- Put all samples on ice and add 2ul Exo I/III mix
- Also add 0.4ul ET SSB to samples 1-6 and 8
- Incubate at 37C for 1hr
- Incubate at 94C for 10min
- qPCR all 16 samples