Daniel:Notebook/ComboLock/2016-11-1: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
(9 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
=Circularization Optimization Testing= | =Circularization Optimization Testing (Started [[Daniel:Notebook/ComboLock/2016-10-31|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
Line 132: | Line 132: | ||
<li>Make master mixes according to following recipes</li> | <li>Make master mixes according to following recipes</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>535.5 uL nfH2O</li> | ||
<li> | <li>637.5 uL SYBR Fast</li> | ||
<li> | <li>25.5 uL 10 uM AmpF</li> | ||
</ol> | </ol> | ||
<li>Add | <li>Add 47 uL master mix to each well</li> | ||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> | ||
<ol type="a"> | <ol type="a"> | ||
Line 150: | Line 150: | ||
<li>TBE Gel</li> | <li>TBE Gel</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix | <li>Mix 240 uL TBE, 60 uL 6x loading dye</li> | ||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | ||
<li>Add 2 uL of sample or ladder to correct drop</li> | <li>Add 2 uL of sample or ladder to correct drop</li> | ||
Line 164: | Line 164: | ||
|- 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="105" height="45" | Sample | | width="105" height="45" | Sample | ||
| width=" | | width="105" | Condition | ||
| width=" | | width="65" | RCA? | ||
| width="65" | Lane | | width="65" | Lane | ||
| width="65" | AmpR Index | | width="65" | AmpR Index | ||
Line 192: | Line 192: | ||
|style="font-weight:bold" align="center" | K-ON-Y | |style="font-weight:bold" align="center" | K-ON-Y | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | A2 | ||
| align="center" align="center" | 20 | | align="center" align="center" | 20 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 205: | Line 205: | ||
|style="font-weight:bold" align="center" | K-ON-N | |style="font-weight:bold" align="center" | K-ON-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | A3 | ||
| align="center" align="center" | 21 | | align="center" align="center" | 21 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 218: | Line 218: | ||
|style="font-weight:bold" align="center" valign="bottom" | K-ON-N | |style="font-weight:bold" align="center" valign="bottom" | K-ON-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | A4 | ||
| align="center" align="center" | 21 | | align="center" align="center" | 21 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 231: | Line 231: | ||
|style="font-weight:bold" align="center" | K-2H-Y | |style="font-weight:bold" align="center" | K-2H-Y | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | A5 | ||
| align="center" align="center" | 22 | | align="center" align="center" | 22 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 244: | Line 244: | ||
|style="font-weight:bold" align="center" | K-2H-Y | |style="font-weight:bold" align="center" | K-2H-Y | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | A6 | ||
| align="center" align="center" | 22 | | align="center" align="center" | 22 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 257: | Line 257: | ||
|style="font-weight:bold" align="center" | K-2H-N | |style="font-weight:bold" align="center" | K-2H-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | A7 | ||
| align="center" align="center" | 23 | | align="center" align="center" | 23 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 270: | Line 270: | ||
|style="font-weight:bold" align="center" valign="bottom" | K-2H-N | |style="font-weight:bold" align="center" valign="bottom" | K-2H-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | A8 | ||
| align="center" align="center" | 23 | | align="center" align="center" | 23 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 283: | Line 283: | ||
|style="font-weight:bold" align="center" | P-ON-Y | |style="font-weight:bold" align="center" | P-ON-Y | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F1 | ||
| align="center" align="center" | 24 | | align="center" align="center" | 24 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 296: | Line 296: | ||
|style="font-weight:bold" align="center" | P-ON-Y | |style="font-weight:bold" align="center" | P-ON-Y | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F2 | ||
| align="center" align="center" | 24 | | align="center" align="center" | 24 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 309: | Line 309: | ||
|style="font-weight:bold" align="center" | P-ON-N | |style="font-weight:bold" align="center" | P-ON-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F3 | ||
| align="center" align="center" | 25 | | align="center" align="center" | 25 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 322: | Line 322: | ||
|style="font-weight:bold" align="center" valign="bottom" | P-ON-N | |style="font-weight:bold" align="center" valign="bottom" | P-ON-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F4 | ||
| align="center" align="center" | 25 | | align="center" align="center" | 25 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 335: | Line 335: | ||
|style="font-weight:bold" align="center" | P-2H-Y | |style="font-weight:bold" align="center" | P-2H-Y | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F5 | ||
| align="center" align="center" | 26 | | align="center" align="center" | 26 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 348: | Line 348: | ||
|style="font-weight:bold" align="center" | P-2H-Y | |style="font-weight:bold" align="center" | P-2H-Y | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F6 | ||
| align="center" align="center" | 26 | | align="center" align="center" | 26 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 361: | Line 361: | ||
|style="font-weight:bold" align="center" | P-2H-N | |style="font-weight:bold" align="center" | P-2H-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F7 | ||
| align="center" align="center" | 27 | | align="center" align="center" | 27 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 374: | Line 374: | ||
|style="font-weight:bold" align="center" valign="bottom" | P-2H-N | |style="font-weight:bold" align="center" valign="bottom" | P-2H-N | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | F8 | ||
| align="center" align="center" | 27 | | align="center" align="center" | 27 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 387: | Line 387: | ||
|style="font-weight:bold" align="center" | K-ON-Y | |style="font-weight:bold" align="center" | K-ON-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H1 | ||
| align="center" align="center" | 28 | | align="center" align="center" | 28 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 400: | Line 400: | ||
|style="font-weight:bold" align="center" | K-ON-Y | |style="font-weight:bold" align="center" | K-ON-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H2 | ||
| align="center" align="center" | 28 | | align="center" align="center" | 28 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 411: | Line 411: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" | Sample 3A | |style="font-weight:bold" height="15" | Sample 3A | ||
|style="font-weight:bold" align="center" | K- | |style="font-weight:bold" align="center" | K-2H-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H3 | ||
| align="center" align="center" | 29 | | align="center" align="center" | 29 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 424: | Line 424: | ||
|- style="background-color:#C4D79B;font-size:12pt" | |- style="background-color:#C4D79B;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 3B | |style="font-weight:bold" height="15" valign="bottom" | Sample 3B | ||
|style="font-weight:bold" align="center" | K- | |style="font-weight:bold" align="center" | K-2H-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H4 | ||
| align="center" align="center" | 29 | | align="center" align="center" | 29 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 439: | Line 439: | ||
|style="font-weight:bold" align="center" | P-ON-Y | |style="font-weight:bold" align="center" | P-ON-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H5 | ||
| align="center" align="center" | 30 | | align="center" align="center" | 30 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 452: | Line 452: | ||
|style="font-weight:bold" align="center" | P-ON-Y | |style="font-weight:bold" align="center" | P-ON-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H6 | ||
| align="center" align="center" | 30 | | align="center" align="center" | 30 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 463: | Line 463: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" | Sample 7A | |style="font-weight:bold" height="15" | Sample 7A | ||
|style="font-weight:bold" align="center" | P- | |style="font-weight:bold" align="center" | P-2H-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H7 | ||
| align="center" align="center" | 31 | | align="center" align="center" | 31 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 476: | Line 476: | ||
|- style="background-color:#C4D79B;font-size:12pt" | |- style="background-color:#C4D79B;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 7B | |style="font-weight:bold" height="15" valign="bottom" | Sample 7B | ||
|style="font-weight:bold" align="center" | P- | |style="font-weight:bold" align="center" | P-2H-Y | ||
| align="center" | Y | | align="center" | Y | ||
| align="center" | | | align="center" | H8 | ||
| align="center" align="center" | 31 | | align="center" align="center" | 31 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 491: | Line 491: | ||
|style="font-weight:bold" align="center" | NA | |style="font-weight:bold" align="center" | NA | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | C1 | ||
| align="center" align="center" | 33 | | align="center" align="center" | 33 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 504: | Line 504: | ||
===Results=== | ===Results=== | ||
<gallery perrow= | <gallery perrow=3 heights=300px widths=300px> | ||
File:|qPCR | File:20161101-qPCR-Circularization-KvsP.png|Klentaq (Blue) vs Phusion (Red) | ||
File:|Gel image | File:20161101-qPCR-Circularization-IncTime.png|Incubation Time; Overnight (Blue) vs 2 Hour (Red) | ||
File:20161101-qPCR-Circularization-RCACompare.png|Rolling Circle Amplification (3hr); Before (Red) and After (Blue) | |||
File:2016-11-01-CircularizationTest-Klentaq.png|Gel image Post-Klentaq | |||
File:2016-11-01-CircularizationTest-Phusion.png|Gel image Post-Phusion | |||
File:2016-11-01-CircularizationTest-PostRCA.png|Gel image Post-RCA | |||
File:2016-11-01-CircularizationTest-PostRCA-Edit.png|Gel image Post-RCA; Lines edit | |||
</gallery> | </gallery> | ||
[[Category:ComboLock]] [[Category:20161031]] |
Latest revision as of 22:20, 2 November 2016
Circularization Optimization Testing (Started Yesterday)[edit]
Sample Matrix[edit]
Sample | Polymerase | Incubation Time | RCA Y/N |
Sample 1 (AB) | Klentaq | ON | Y |
Sample 2 (AB) | Klentaq | ON | N |
Sample 3 (AB) | Klentaq | 2 hr | Y |
Sample 4 (AB) | Klentaq | 2 hr | N |
Sample 5 (AB) | Phusion | ON | Y |
Sample 6 (AB) | Phusion | ON | N |
Sample 7 (AB) | Phusion | 2 hr | Y |
Sample 8 (AB) | Phusion | 2 hr | N |
Protocol[edit]
- Circularization (all samples)
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 30 uL exonuclease I (20 units/μL) and 6 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- Rolling Circle Amplification (Samples 1,3,5,7; Hold samples 2,4,6,8 at 4C)
- Prepare 8.1X Master mix according to table below
- 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
- qPCR
- Set up reaction according to table below
- Make master mixes according to following recipes
- 535.5 uL nfH2O
- 637.5 uL SYBR Fast
- 25.5 uL 10 uM AmpF
- Add 47 uL master mix to each well
- 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 240 uL TBE, 60 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
Reagent | uL Added | Master Mix (8.1X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 20.25 |
dNTP (1 mM) | 0.8 | 6.48 |
10X Buffer | 2 | 16.2 |
Phi29 | 1 | 8.1 |
nfH2O | 8.7 | 70.47 |
Total | 20 | 121.5 |
qPCR Sample Table[edit]
Sample | Condition | RCA? | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | K-ON-Y | N | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | K-ON-Y | N | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | K-ON-N | N | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | K-ON-N | N | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | K-2H-Y | N | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | K-2H-Y | N | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | K-2H-N | N | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | K-2H-N | N | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | P-ON-Y | N | F1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | P-ON-Y | N | F2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6A | P-ON-N | N | F3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6B | P-ON-N | N | F4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7A | P-2H-Y | N | F5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7B | P-2H-Y | N | F6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 8A | P-2H-N | N | F7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 8B | P-2H-N | N | F8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | K-ON-Y | Y | H1 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | K-ON-Y | Y | H2 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | K-2H-Y | Y | H3 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | K-2H-Y | Y | H4 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5A | P-ON-Y | Y | H5 | 30 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5B | P-ON-Y | Y | H6 | 30 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7A | P-2H-Y | Y | H7 | 31 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 7B | P-2H-Y | Y | H8 | 31 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | NA | N | C1 | 33 | 2 | 25 | 1 | 1 | 21 | 50 |
Results[edit]
- 20161101-qPCR-Circularization-KvsP.png
Klentaq (Blue) vs Phusion (Red)
- 20161101-qPCR-Circularization-IncTime.png
Incubation Time; Overnight (Blue) vs 2 Hour (Red)
- 20161101-qPCR-Circularization-RCACompare.png
Rolling Circle Amplification (3hr); Before (Red) and After (Blue)
- 2016-11-01-CircularizationTest-Klentaq.png
Gel image Post-Klentaq
- 2016-11-01-CircularizationTest-Phusion.png
Gel image Post-Phusion
- 2016-11-01-CircularizationTest-PostRCA.png
Gel image Post-RCA
- 2016-11-01-CircularizationTest-PostRCA-Edit.png
Gel image Post-RCA; Lines edit