Matt:LabNotes/2016-11-15: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (Created page with "=In vitro capture with CA12kOct2016 V4 SplintR Ligase Test 2= *gDNA and UHRR *SplintR and T4 DNA Ligase for HBRR ==Reference== *[[Matt:LabNotes/2016-9-9#Results|SplintR in vi...") |
>Mzcai mNo edit summary |
||
Line 14: | Line 14: | ||
===Sample Groups=== | ===Sample Groups=== | ||
*RNA template = | *RNA template = 100ng Universal Human Reference RNA (UHRR 740000-41) | ||
*DNA template = 300ng gDNA 12878 | *DNA template = 300ng gDNA 12878 | ||
#NTC - | #NTC - 9.9ng V4 - SplintR | ||
# | #150ng RNA - 9.9ng V4 - SplintR | ||
#150ng RNA - | #150ng RNA - 9.9ng V4 - T4 | ||
# | #160ng gDNA - 9.9ng V4 - Ampligase | ||
==Experiment== | ==Experiment== | ||
===V4 Padlock Probe Capture=== | ===V4 Padlock Probe Capture=== | ||
*Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc) | *Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc) | ||
*DNA is 12878 80.3ng/ul | *DNA is 12878 80.3ng/ul | ||
* | *V4 padlock probes: *[[Matt:LabNotes/2016-11-2|20.7nM]] | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="font-size:11pt" valign="bottom" | |- style="font-size:11pt" valign="bottom" | ||
| width="51" height="14" | Sample | | width="51" height="14" | Sample | ||
| width="51" | RNA (50ng/ul) | | width="51" | RNA (50ng/ul) | ||
| width="51" | DNA (80.3ng/ul) | | width="51" | DNA (80.3ng/ul) | ||
| width="51" | | | width="51" | V4 | ||
| width="51" | 10X Buffer | |||
| width="51" | 10X | |||
| width="51" | H2O | | width="51" | H2O | ||
| width="51" | Total | | width="51" | Total | ||
|- style="font-size:11pt" valign="bottom" | |- style="font-size:11pt" valign="bottom" | ||
| | | height="14" | NTC | ||
| align="right" | 0 | | align="right" | 0 | ||
| align="right" | 0 | | align="right" | 0 | ||
| align="right" | | | align="right" | 10 | ||
| align="right" | 3 | | align="right" | 3 | ||
| align="right" | | | align="right" | 17 | ||
| align="right" | 30 | | align="right" | 30 | ||
|- style="font-size:11pt" valign="bottom" | |- style="font-size:11pt" valign="bottom" | ||
| | | height="14" | SplintR | ||
| align="right" | 3 | | align="right" | 3 | ||
| align="right" | 0 | | align="right" | 0 | ||
| align="right" | | | align="right" | 10 | ||
| align="right" | 3 | | align="right" | 3 | ||
| align="right" | | | align="right" | 14 | ||
| align="right" | 30 | | align="right" | 30 | ||
|- style="font-size:11pt" valign="bottom" | |- style="font-size:11pt" valign="bottom" | ||
| | | height="14" | T4 | ||
| align="right" | 3 | | align="right" | 3 | ||
| align="right" | 0 | | align="right" | 0 | ||
| align="right" | | | align="right" | 10 | ||
| align="right" | 3 | | align="right" | 3 | ||
| align="right" | | | align="right" | 14 | ||
| align="right" | 30 | | align="right" | 30 | ||
|- style="font-size:11pt" valign="bottom" | |- style="font-size:11pt" valign="bottom" | ||
| | | height="14" | Ampligase | ||
| align="right" | 0 | | align="right" | 0 | ||
| align="right" | 2 | | align="right" | 2 | ||
| align="right" | 10 | |||
| align="right" | | |||
| align="right" | 3 | | align="right" | 3 | ||
| align="right" | | | align="right" | 15 | ||
| align="right" | 30 | | align="right" | 30 | ||
|} | |} | ||
*Added 50ul mineral oil on top | *Added 50ul mineral oil on top | ||
'''Program'''<br> | '''Program'''<br> | ||
* 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h | * 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! | *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: | ||
<!-- | |||
* | |||
===qPCR=== | ===qPCR=== | ||
====Primers==== | ====Primers==== |
Revision as of 23:55, 16 November 2016
In vitro capture with CA12kOct2016 V4 SplintR Ligase Test 2
- gDNA and UHRR
- SplintR and T4 DNA Ligase for HBRR
Reference
Experimental Outline
- CA12kOct2016 V4 Padlock Probe capture of gDNA and RNA
- Sequence to quantify probe efficiencies
Sample Groups
- RNA template = 100ng Universal Human Reference RNA (UHRR 740000-41)
- DNA template = 300ng gDNA 12878
- NTC - 9.9ng V4 - SplintR
- 150ng RNA - 9.9ng V4 - SplintR
- 150ng RNA - 9.9ng V4 - T4
- 160ng gDNA - 9.9ng V4 - Ampligase
Experiment
V4 Padlock Probe Capture
- 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: