Daniel:Notebook/ComboLock/2017-1-25: Difference between revisions
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Still have some left, so I'm going to test its effectiveness by trying to amplify the 1 amol sample. I'll use the original Epicentre concentration and a 10X dilution, since the Epicentre Phi29 is 10X more concentrated than NEB. | Still have some left, so I'm going to test its effectiveness by trying to amplify the 1 amol sample. I'll use the original Epicentre concentration and a 10X dilution, since the Epicentre Phi29 is 10X more concentrated than NEB. | ||
'''Sample Matrix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center" | |||
| width="100" height="32" | Sample | |||
| width="95" | Condition | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6AC | |||
| align="center" valign="bottom" | Epicentre-1:10 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6BC | |||
| align="center" valign="bottom" | Epicentre-1:10 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6AD | |||
| align="center" valign="bottom" | Epicentre | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6BD | |||
| align="center" valign="bottom" | Epicentre | |||
|} | |||
<ol> | <ol> | ||
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{| 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="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="250" height="30" | Reagent | ||
| width="145" | uL Added | | width="145" | uL Added | ||
| width=" | | width="105" | Master Mix (4.5X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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<li>Incubate at 37C for 3 hours</li> | <li>Incubate at 37C for 3 hours</li> | ||
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | ||
Held overnight at 4C; Continued [[Daniel:Notebook/ComboLock/2017-1-26|tomorrow]] | |||
==Library Prep== | ==Library Prep== | ||
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Based on [[Daniel:Notebook/ComboLock/2017-1-24|yesterday]]'s results, I'm going to sequence the most successful lanes (PreRCA-1pmol; PostRCA-1 pmol, 10 fmol, and 100 amol). With the UMI now on the padlock, this should give a relative percentage of padlock capture. Each sample has a unique AmpR index. | Based on [[Daniel:Notebook/ComboLock/2017-1-24|yesterday]]'s results, I'm going to sequence the most successful lanes (PreRCA-1pmol; PostRCA-1 pmol, 10 fmol, and 100 amol). With the UMI now on the padlock, this should give a relative percentage of padlock capture. Each sample has a unique AmpR index. | ||
'''Sample Matrix''' | |||
{| 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="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="120" height="32" | Sample | ||
| width=" | | width="90" | Sample Amt | ||
| width=" | | width="70" | Pre/Post RCA | ||
| width=" | | width="90" | AmpR Index | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
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|} | |} | ||
<ol> | <ol> | ||
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<li>Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)</li> | <li>Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)</li> | ||
<li>Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube</li> | <li>Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube</li> | ||
<li>Aliquot 24 | <li>Aliquot 48/24 uL per sample/ladder lane into each well</li> | ||
<li>Run gel for 23 minutes at 230V</li> | <li>Run gel for 23 minutes at 230V</li> | ||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | ||
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<li>Image in gel doc</li> | <li>Image in gel doc</li> | ||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> | ||
File:|Before Image | File:2017-01-25-RCATest-SizeSelect.png|Before Image | ||
File:|After Image | File:2017-01-25-RCATest-SizeSelect-After.png|After Image | ||
</gallery> | </gallery> | ||
<li>Centrifuge at 12000 rpm for 1.5 minutes</li> | <li>Centrifuge at 12000 rpm for 1.5 minutes</li> | ||
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<ol type="A"> | <ol type="A"> | ||
<li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample</li> | <li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample</li> | ||
<li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2017-1- | <li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2017-1-26|tomorrow]]</li> | ||
</ol></ol> | </ol></ol> | ||
[[Category:ComboLock]] [[Category:20170123]] | [[Category:ComboLock]] [[Category:20170123]] |
Latest revision as of 19:03, 16 February 2017
Circularization Test (Started Monday)[edit]
Protocol-New Amplification Primers[edit]
These are new primers I designed. They are slightly adjusted from the locations of AmpF/AmpR. Hopefully there is less noise.
- qPCR
- Make qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 17.2 uL AmpF-CLv3
- 17.2 uL AmpR-CLv3
- 430 uL Kapa SYBR Fast
- Add 48 uL master mix to each well
- Add 2 uL sample according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 80 uL TBE, 20 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Product Amt | Pre/Post RCA | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 0A | 1pmol | Pre | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0B | 1pmol | Pre | A2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | 10 fmol | Pre | A3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | 10 fmol | Pre | A4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | 100 amol | Pre | A5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | 100 amol | Pre | A6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6A | 1 amol | Pre | A7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6B | 1 amol | Pre | A8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0AX | 1pmol | Post | H1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0BX | 1pmol | Post | H2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AX | 10 fmol | Post | H3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BX | 10 fmol | Post | H4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AX | 100 amol | Post | H5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BX | 100 amol | Post | H6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6AX | 1 amol | Post | H7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BX | 1 amol | Post | H8 | 2 | 25 | 1 | 1 | 21 | 50 |
Results[edit]
- 20170125-qPCR-NewAmp.png
qPCR Curve
- 2017-01-25-RCATest-NewAmp-Pre.png
Gel image-Pre RCA
- 2017-01-25-RCATest-NewAmp-Post.png
Gel image-Post RCA
RCA with Epicentre Phi29[edit]
Still have some left, so I'm going to test its effectiveness by trying to amplify the 1 amol sample. I'll use the original Epicentre concentration and a 10X dilution, since the Epicentre Phi29 is 10X more concentrated than NEB.
Sample Matrix
Sample | Condition |
Sample 6AC | Epicentre-1:10 |
Sample 6BC | Epicentre-1:10 |
Sample 6AD | Epicentre |
Sample 6BD | Epicentre |
- Rolling Circle Amplification
- Prepare 7X 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 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- Size Select Gel
- Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)
- Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
- Aliquot 48/24 uL per sample/ladder lane into each well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Image in gel doc
- 2017-01-25-RCATest-SizeSelect.png
Before Image
- 2017-01-25-RCATest-SizeSelect-After.png
After Image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate 4 hours at 37C with vigorous shaking;
- Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
- Extract supernatant and add to nanosep column
- Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
- Incubate at -80C overnight; continued tomorrow
Reagent | uL Added | Master Mix (4.5X) |
Template | 5 | NA |
RCA Primer (LLRC 10 uM) | 2.5 | 11.25 |
dNTP (1 mM) | 0.8 | 3.6 |
10X Buffer | 2 | 9 |
Phi29 | 1 | 0 |
nfH2O | 8.7 | 39.15 |
Total | 20 | 63 |
Held overnight at 4C; Continued tomorrow
Library Prep[edit]
Based on yesterday's results, I'm going to sequence the most successful lanes (PreRCA-1pmol; PostRCA-1 pmol, 10 fmol, and 100 amol). With the UMI now on the padlock, this should give a relative percentage of padlock capture. Each sample has a unique AmpR index.
Sample Matrix
Sample | Sample Amt | Pre/Post RCA | AmpR Index |
Sample 0 (AB) | 1 pmol | Pre | 21 |
Sample 0X (AB) | 1 pmol | Post | 25 |
Sample 2X (AB) | 10 fmol | Post | 26 |
Sample 4X (AB) | 100 amol | Post | 27 |