Matt:LabNotes/2016-11-15: Difference between revisions

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====PCR Test for Non-Enzyme Digested Samples====
====PCR Test====
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 128: Line 128:
   Program
   Program
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min
<!--
[[File:20161119_CA12kOct2016V4_invitroCapture_PCRtest.JPG|450px]]
==Results==
===Before Exo I/III Digest of Unligated Padlock Probes===
[[File:20160909_SplintRTest2_qPCR_NotDigested.JPG|350px]][[File:20160909_SplintRTest2_qPCR_Digested.JPG|350px]]
====Template Digested Notes====
*Biggest factor for ligated products is concentration of padlock probes added
**Suggests there is a lot of non-specific annealing between padlock probes at higher concentrations
*Separation between NTC and RNA template samples increases as padlock probe concentration decreases
**30ng samples looked best, with NTC less than NegCtrl
*PosCtrl less ligated products than NegCtrl is an anomaly
**Probably cause: NegCtrl had 2.5X more padlock probes added than PosCtrl
**15min of Ampligase is not enough time for PosCtrl to have a lot of ligation compared to normal protocol of 24hrs


====Template Not Digested Notes====
====PCR====
*Theoretically should be the same? Since RNA template cannot be amplified by KAPA polymerase and thermocycling should denature padlock probes from template
{| {{table}}
**Maybe PCR primers are occupied with template
| align="center" style="background:#f0f0f0;"|'''Components'''
 
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
*Separation between sample and NTC for 820ng and 30ng almost non-existent but 150ng still has good separation
| align="center" style="background:#f0f0f0;"|'''3.5X Volume'''
**I could see more template leads to PCR primers being occupied on template, which is why 820ng sample amplifies less than NTC but I would then expect 150ng to show more similarity to that than 30ng
|-
*PosCtrl and NegCtrl look "correct"
| Captured template||12||0
===After Exo I/III Digest of Unligated Padlock Probes===
|-
[[File:20160912_SplintRTest2_qPCR_NotDigested.JPG|350px]][[File:20160912_SplintRTest2_qPCR_Digested.JPG|350px]]
| 10uM Forward Primer||2||7
====Template Digested Notes====
|-
*This is the "correct" experiment
| 10uM Reverse Primer||2||0
**Everything points to using less padlock probes limits false positives in NTC
|-
*SplintR is much more active than Ampligase
| 2X KAPA SYBG MM||50||175
====Template Not Digested Notes====
|-
*Oddly this shows the ~30nM Padlock Probe concentration of 150ng samples is the sweet spot
| H2O||34||119
**No idea why there is such a large separation if RNA is not digested
|-
 
| Total||100||301
==Conclusion==
|}
*56.4C annealing temperature might be too low
*Indx1:T4
*Ligase time of 15min might be too long at 37C
*Indx2:SplintR
*Biggest factor for ligated products is concentration of padlock probes
*Indx3:Ampligase
 
*Aliquot 86ul from 3.5X master mix and add 12ul captured template and 2ul corresponding reverse primer
 
  Program
*Try in situ, hopefully false positive is not an issue by washing away non-annealed padlock probes
  98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
*Need to use a stringent wash buffer (30% formamide)?
[[File:20161203_invitro_CA12kOct2016V4_PCR.png|450px]]


====AMPure Bead Purification====
*1:1 beads to amplicon volume ratio
**Eluted with 50ul total each sample


-->
====PAGE Check====
*Load 2ul sample + 2ul loading dye
[[File:2016-12-15_CA12kOct2016_V4_invitroCapture_Library.jpg|250px]]
====Sequencing Library====
MC20161215_CA12kOct20161215_V4_T4_Indx1<br>
MC20161215_CA12kOct20161215_V4_SplintR_Indx2<br>
MC20161215_CA12kOct20161215_V4_Amp_Indx3

Latest revision as of 01:55, 16 December 2016

In vitro capture with CA12kOct2016 V4[edit]

  • gDNA and UHRR
  • SplintR and T4 DNA Ligase for HBRR

Reference[edit]

Experimental Outline[edit]

  1. CA12kOct2016 V4 Padlock Probe capture of gDNA and RNA
  2. Sequence to quantify probe efficiencies

Sample Groups[edit]

  • RNA template = 100ng Universal Human Reference RNA (UHRR 740000-41)
  • DNA template = 300ng gDNA 12878
  1. NTC - 9.9ng V4 - SplintR
  2. 150ng RNA - 9.9ng V4 - SplintR
  3. 150ng RNA - 9.9ng V4 - T4
  4. 160ng gDNA - 9.9ng V4 - Ampligase

Experiment[edit]

V4 Padlock Probe Capture[edit]

  • Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc)
  • DNA is 12878 80.3ng/ul
  • V4 padlock probes: *20.7nM
Sample RNA (50ng/ul) DNA (80.3ng/ul) V4 10X Buffer H2O Total
NTC 0 0 10 3 17 30
SplintR 3 0 10 3 14 30
T4 3 0 10 3 14 30
Ampligase 0 2 10 3 15 30
  • Added 50ul mineral oil on top

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • -> add 3ul Enzyme mix
  • For Ampligase: -> 55C 20h -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold
  • For SplintR and T4: -> 37C 15min -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold
  • Add 3ul Enzyme mix prepared on ice!
    • Ampligase: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
    • NTC & SplintR: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O
    • T4: 1ul T4 DNA Ligase + 1ul 10X T4 Buffer + 5.5ul ATP + 2.5ul H2O
      • Thermo T4 DNA Ligase HC, 30 Weiss U/ul
      • ATP 10mM

qPCR[edit]

Primers[edit]

Primer Sequence Index #
ISB_CA_AF AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG
ISB_CA_AR.T1 CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG Indx1
ISB_CA_AR.T2 CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG Indx2
ISB_CA_AR.T3 CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG Indx3

PCR Test[edit]

Components 1X Volume 4X Volume
Captured template 1 0
10uM ISB_CA_AF 0.4 1.6
10uM ISB_CA_AR.T1 0.4 1.6
2X KAPA SYBG MM 12.5 50
H2O 10.7 42.8
Total 25 96
  • Aliquot 24ul from 4X master mix and add 1ul captured template
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min

File:20161119 CA12kOct2016V4 invitroCapture PCRtest.JPG

PCR[edit]

Components 1X Volume 3.5X Volume
Captured template 12 0
10uM Forward Primer 2 7
10uM Reverse Primer 2 0
2X KAPA SYBG MM 50 175
H2O 34 119
Total 100 301
  • Indx1:T4
  • Indx2:SplintR
  • Indx3:Ampligase
  • Aliquot 86ul from 3.5X master mix and add 12ul captured template and 2ul corresponding reverse primer
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

File:20161203 invitro CA12kOct2016V4 PCR.png

AMPure Bead Purification[edit]

  • 1:1 beads to amplicon volume ratio
    • Eluted with 50ul total each sample

PAGE Check[edit]

  • Load 2ul sample + 2ul loading dye

File:2016-12-15 CA12kOct2016 V4 invitroCapture Library.jpg

Sequencing Library[edit]

MC20161215_CA12kOct20161215_V4_T4_Indx1
MC20161215_CA12kOct20161215_V4_SplintR_Indx2
MC20161215_CA12kOct20161215_V4_Amp_Indx3