Editing
Matt:LabNotes/2016-9-9
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=SplintR Ligase Test 2 = *[[Matt:LabNotes/2016-8-21|Previous test]] ==Reference== *[[Matt:LabNotes/2015-1-21|V4 and V7 Capture Experiment]] *[[Matt:LabNotes/2015-3-19|V4 Capture Analysis]] *[[Matt:LabNotes/2015-1-26|V6 & V8 Probe Production]] ==Experimental Outline== #V8 Padlock Probe capture to RNA #*Ran out of V6 probes used last time #*V8 is a subset of V6 probes (targets constitutive exons instead of contigs of exons) #Quantify captured products by qPCR #*Also adds Illumina sequencing adapters ===Sample Groups=== *Use Universal Human Reference RNA (UHRR 740000-41) *3 RNA template concentrations for each (30ng, 150ng and 820ng) **For DNA template positive control only do 1 sample using 300ng gDNA 12878 #NTC - 8.19ng V8 - SplintR #NTC - 40.9ng V8 - SplintR #NTC - 221.1ng V8 - SplintR #30ng RNA - 8.19ng V8 - SplintR #150ng RNA - 40.9ng V8 - SplintR #820ng RNA - 221.1ng V8 - SplintR #PosCtrl: 300ng DNA - 16.38ng V8 - Ampligase #NegCtrl: 150ng RNA - 40.9ng V8 - Ampligase *Summary: **3 NTC samples with varying amount of padlock probes that matches experimental sample **3 experimental samples of 30ng, 150ng, and 820ng UHRR **1 PosCtrl that uses Ampligase for V8 to capture 300ng gDNA (876:1 probe:target ratio) **1 NegCtrl that uses Ampligase for V8 to capture 150ng UHRR ==Experiment== ===V8 Padlock Probe Capture=== *Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc) *DNA is 12878 80.3ng/ul *V8 padlock probes: [[Matt:LabNotes/2015-1-21|874nM]] **Add 5.2ul to samples 3 & 6 and then dilute remaining ~4ul to 19ul final volume {| 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" | RNA (50ng/ul) | width="51" | DNA (80.3ng/ul) | width="51" | V8 (42.7ng/ul) | width="51" | V8 (8.19ng/ul) | width="51" | 10X SplintR Buffer (*=Ampligase) | width="51" | H2O | width="51" | Total |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 1 | align="right" | 3 | align="right" | 26 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 2 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 5 | align="right" | 3 | align="right" | 22 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 5.2 | align="right" | 0 | align="right" | 3 | align="right" | 21.8 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 4 | align="right" | 0.6 | align="right" | 0 | align="right" | 0 | align="right" | 1 | align="right" | 3 | align="right" | 25.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 5 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 5 | align="right" | 3 | align="right" | 19 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 6 | align="right" | 16.4 | align="right" | 0 | align="right" | 5.2 | align="right" | 0 | align="right" | 3 | align="right" | 5.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 7 | align="right" | 0 | align="right" | 3.8 | align="right" | 0 | align="right" | 2 | 3* | align="right" | 21.2 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 8 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 5 | 3* | align="right" | 19 | align="right" | 30 |} *Added 50ul mineral oil on top '''Program'''<br> * 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h *Add 3ul Enzyme mix prepared on ice! **Samples 7-8: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O **Samples 1-6: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O *Incubate at 37C for 15min *94C for 2min *Aliquot 10ul from each into separate strip of tubes and put at 4C for 1 hr until qPCR *Enzyme digest template of remaining mixture 37C for 1 hr **1ul Exo I/III (1:1) for DNA **1ul RNaseH and Riboshredder (1:1) for RNA ***First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder) *94C for 2min *Add 1ul Exo I/III (1:1) for unligated padlock probes to ALL tubes *37C for 1 hr *94C for 2min ===qPCR=== ====Primers==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Primer''' | align="center" style="background:#f0f0f0;"|'''Sequence''' | align="center" style="background:#f0f0f0;"|'''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 for Non-Enzyme Digested Samples==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''16X Volume''' |- | Captured template||1||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||10.7||171.2 |- | Total||25||384 |} *Aliquot 24ul from 18X master mix and add 1ul captured template Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min ==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==== *Theoretically should be the same? Since RNA template cannot be amplified by KAPA polymerase and thermocycling should denature padlock probes from template **Maybe PCR primers are occupied with template *Separation between sample and NTC for 820ng and 30ng almost non-existent but 150ng still has good separation **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" ===After Exo I/III Digest of Unligated Padlock Probes=== [[File:20160912_SplintRTest2_qPCR_NotDigested.JPG|350px]][[File:20160912_SplintRTest2_qPCR_Digested.JPG|350px]] ====Template Digested Notes==== *This is the "correct" experiment **Everything points to using less padlock probes limits false positives in NTC *SplintR is much more active than Ampligase ====Template Not Digested Notes==== *Oddly this shows the ~30nM Padlock Probe concentration of 150ng samples is the sweet spot **No idea why there is such a large separation if RNA is not digested ==Conclusion== *56.4C annealing temperature might be too low *Ligase time of 15min might be too long at 37C *Biggest factor for ligated products is concentration of padlock probes *Try in situ, hopefully false positive is not an issue by washing away non-annealed padlock probes *Need to use a stringent wash buffer (30% formamide)?
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information