Daniel:Notebook/ComboLock/2017-1-20: Difference between revisions
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===Results=== | ===Results=== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="130" height="36" | Sample Name | |||
| width="65" | 1A | |||
| width="65" | 1B | |||
| width="65" | 1AL | |||
| width="65" | 1BL | |||
| width="65" | 4AL | |||
| width="65" | 4BL | |||
|- style="font-size:12pt" | |||
| height="30" | Sample Condition | |||
| align="center" | Sample-PreRCA | |||
| align="center" | Sample-PreRCA | |||
| align="center" | Sample | |||
| align="center" | Sample | |||
| align="center" | No PO4 (PCCB) | |||
| align="center" | No PO4 (PCCB) | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" | RCA Primer | |||
| align="center" | None | |||
| align="center" | None | |||
| align="center" | Random Hexamer | |||
| align="center" | Random Hexamer | |||
| align="center" | Random Hexamer | |||
| align="center" | Random Hexamer | |||
|- style="font-size:12pt" | |||
| height="15" | Conc (ng/uL) | |||
| align="center" align="center" | 0.81 | |||
| align="center" align="center" | 1.27 | |||
| align="center" align="center" | 16.6 | |||
| align="center" align="center" | 16.5 | |||
| align="center" align="center" | 15.4 | |||
| align="center" align="center" | 20.5 | |||
|- style="font-size:12pt" | |||
| height="30" valign="bottom" | Fold Change from Pre-RCA | |||
| align="center" | NA | |||
| align="center" | NA | |||
| align="center" align="center" | 20.5 | |||
| align="center" align="center" | 13.0 | |||
| align="center" align="center" | 19.0 | |||
| align="center" align="center" | 16.1 | |||
|} | |||
[[Category:ComboLock]] [[Category:20170113]] | [[Category:ComboLock]] [[Category:20170113]] |
Revision as of 22:37, 20 January 2017
Version 3 Oligos (Started Thursday, Jan 13)
RCA Test-Adpaters
From yesterday's results I'm going to try the short RCA again, this time using the adapter regions as primers. I'll test using Lock0301 and Latch0301, since those should only amplify if circular product exists.
- Rolling Circle Amplification
- Prepare 8.5X Master mix according to table below
- Add 12.5 uL master mix to each tube
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 30 minutes
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- Qubit Measure
- Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)
- Add 190 uL dye/buffer and 10 uL standard to standard tubes
- Add 199 uL dye/buffer and 1 uL sample to sample tubes
- Measure in Qubit
Reagent | uL Added | Master Mix (8.5X) |
Template | 5 | NA |
RCA Primer (Latch0301 or Lock0301); 10 uM) | 2.5 | 21.25 |
dNTP (1 mM) | 0.8 | 6.8 |
10X Phi29 Buffer | 2 | 17 |
Phi29 | 1 | 8.5 |
nfH2O | 8.7 | 73.95 |
Total | 20 | 127.5 |
Results
Sample Name | 1A | 1B | 1AL | 1BL | 4AL | 4BL | 1AK | 1BK | 4AK | 4BK |
Sample Condition | Sample-PreRCA | Sample-PreRCA | Sample | Sample | No PO4 (PCCB) | No PO4 (PCCB) | Sample | Sample | No PO4 (PCCB) | No PO4 (PCCB) |
RCA Primer | None | None | Latch0301 | Latch0301 | Latch0301 | Latch0301 | Primer6 | Primer6 | Primer6 | Primer6 |
Conc (ng/uL) | 0.81 | 1.27 | 1.76 | 2.52 | 2.22 | 2.35 | 0.25 | 0.27 | 0.37 | 0.29 |
Fold Change from Pre-RCA | NA | NA | 2.2 | 2.0 | 2.7 | 1.9 | 0.3 | 0.2 | 0.5 | 0.2 |
So this doesn't show anything definitive. It seems that the RCA didn't work at all with either of these. I'm going to try hexamers, which ought to amplify anything and everything (exponentially, actually)
Random Hexamer RCA
RCA Test (again) using random hexamers.
- Rolling Circle Amplification
- Prepare 4.5X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 30 minutes
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- Qubit Measure
- Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)
- Add 190 uL dye/buffer and 10 uL standard to standard tubes
- Add 199 uL dye/buffer and 1 uL sample to sample tubes
- Wait 5 minutes to allow dye saturation
- Measure in Qubit
Reagent | uL Added | Master Mix (4.5X) |
Template | 5 | NA |
Random Hexamer (100 uM) | 2.5 | 11.25 |
dNTP (1 mM) | 0.8 | 3.6 |
10X Phi29 Buffer | 2 | 9 |
Phi29 | 1 | 4.5 |
nfH2O | 8.7 | 39.15 |
Total | 20 | 67.5 |
Results
Sample Name | 1A | 1B | 1AL | 1BL | 4AL | 4BL |
Sample Condition | Sample-PreRCA | Sample-PreRCA | Sample | Sample | No PO4 (PCCB) | No PO4 (PCCB) |
RCA Primer | None | None | Random Hexamer | Random Hexamer | Random Hexamer | Random Hexamer |
Conc (ng/uL) | 0.81 | 1.27 | 16.6 | 16.5 | 15.4 | 20.5 |
Fold Change from Pre-RCA | NA | NA | 20.5 | 13.0 | 19.0 | 16.1 |