Matt:LabNotes/2017-4-22: Difference between revisions

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(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
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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 18: Line 24:
*ppGFAP
*ppGFAP
*ppMALAT1
*ppMALAT1
**Has the MALAT1_template
*Template for ppMALAT1: MALAT1_template


==Protocol==
==Protocol==
#Add 100nM of each padlock probe to hybridization mix containing SplintR buffer + 0%, 10%, 20% or 30% formamide l
#Combine padlock probes and template in 1X Ligase buffer and possibly formamide
#Hybridize at 55C for 13 hours
#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

  1. Standard: SplintR only
  2. SplintR incubated for 30min instead of 15min
  3. SplintR + 10% formamide
  4. SplintR + 20% formamide
  5. SplintR + 30% formamide
  6. SplintR + 250ng ET SSB
  7. SplintR + 250ng ET SSB added first
  8. 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

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

  1. Combine padlock probes and template in 1X Ligase buffer and possibly formamide
  2. Add mineral oil on top
  3. Incubate at 55C for 17hr
  1. To sample 7 (2-step ET SSB) add 200ng (0.4ul) of ET SSB and keep at 55C for 30min
  2. To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min
    • Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
  3. After sample 7 has ET SSB for 30min move samples 1-7 to 37C
  4. 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
  5. Put all samples on ice and add 2ul Exo I/III mix
    • Also add 0.4ul ET SSB to samples 1-5 and 8
  6. Incubate at 37C for 1hr
  7. 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