Daniel:Notebook/ComboLock/2017-3-21: Difference between revisions
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>Djacobse (Created page with "=Circularization Comparison (T4 Poly/Ligase; Started Wednesday)= Back to Calendar ==Bgl Digestion== Be...") |
>Djacobse |
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(8 intermediate revisions by the same user not shown) | |||
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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:#808000;font-size:12pt" align="center" | |- style="background-color:#808000;font-size:12pt" align="center" | ||
| width=" | | width="85" height="30" | Sample Origin | ||
| width=" | | width="220" | Condition | ||
| width=" | | width="95" | Sample Numbers | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | F | | height="15" | F | ||
| T4 Ligase | | T4 Ligase | ||
| align="center" | 4 | | align="center" | 1,4 | ||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |- style="background-color:#BFBFBF;font-size:12pt" align="center" | ||
Line 40: | Line 40: | ||
{| 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:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="200" height="32" | Reagent | ||
| width=" | | width="120" | Single Rxn Vol | ||
| width=" | | width="120" | 14.2X MM uL | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
Line 51: | Line 51: | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Bglii Cutter Primer (10 uM) | | height="15" valign="bottom" | Bglii Cutter Primer (10 uM) | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 5 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 71 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 10X Buffer 3.1 | | height="15" valign="bottom" | 10X Buffer 3.1 | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 28.4 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Bglii | | height="15" valign="bottom" | Bglii | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 7 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 99.4 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 198.8 | ||
|} | |} | ||
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<li>Heat kill with 65C for 20 min</li></ol> | <li>Heat kill with 65C for 20 min</li></ol> | ||
</ol> | </ol> | ||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make qPCR master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>319.2 uL nfH2O</li> | |||
<li>15.2 uL AmpF</li> | |||
<li>15.2 uL AmpR-Ind22</li> | |||
<li>380 uL Kapa SYBR Fast 2X Master Mix</li></ol> | |||
<li>Add 48 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to plate layout</li> | |||
[[Image:PlateLayout-T4Circ-BgliiDigest-20170321.png|600px]] | |||
<li>qPCR Cycles</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>55C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x30</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 120 uL TBE and 40 uL 6X dye</li> | |||
<li>Aliquot 10 uL per sample on to parafilm</li> | |||
<li>Add 2 uL sample or 1.5 uL ladder to aliquots</li> | |||
<li>Mix and add 10 uL per lane to gel</li> | |||
<li>Run gel at 230V for 24 minutes</li> | |||
<li>Add 2 uL SYBR gold and incubate on shaker for 3 minutes</li> | |||
<li>Rinse and image in gel doc</li></ol> | |||
</ol> | |||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:20170321-qPCR-T4Circ-PostBglii.png|qPCR curves | |||
File:2017-03-21-T4Circ-PostBglii-T4Samples.png|Gel image-T4 Ligase samples (F) | |||
File:2017-03-21-T4Circ-PostBglii-APSamples.png|Gel image-Amp/Phusion samples (G,H) | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20170315]] |
Latest revision as of 22:18, 21 March 2017
Circularization Comparison (T4 Poly/Ligase; Started Wednesday)[edit]
Bgl Digestion[edit]
Because I CAN. Also, will determine if there is an improvement in signal post-digestion.
Sample Matrix
Sample Origin | Condition | Sample Numbers |
F | T4 Ligase | 1,4 |
G | Phusion/Amp (Unpurified) | 1,4 |
H | Phusion/AmpHS (Unpurified) | 1,4 |
- Bglii Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 4.2X master mix
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- qPCR
- Make qPCR master mix according to following recipe
- 319.2 uL nfH2O
- 15.2 uL AmpF
- 15.2 uL AmpR-Ind22
- 380 uL Kapa SYBR Fast 2X Master Mix
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-T4Circ-BgliiDigest-20170321.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 120 uL TBE and 40 uL 6X dye
- Aliquot 10 uL per sample on to parafilm
- Add 2 uL sample or 1.5 uL ladder to aliquots
- Mix and add 10 uL per lane to gel
- Run gel at 230V for 24 minutes
- Add 2 uL SYBR gold and incubate on shaker for 3 minutes
- Rinse and image in gel doc
Reagent | Single Rxn Vol | 14.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 5 | 71 |
10X Buffer 3.1 | 2 | 28.4 |
Bglii | 1 | 0 |
nfH2O | 7 | 99.4 |
Total | 20 | 198.8 |
Results[edit]
- 20170321-qPCR-T4Circ-PostBglii.png
qPCR curves
- 2017-03-21-T4Circ-PostBglii-T4Samples.png
Gel image-T4 Ligase samples (F)
- 2017-03-21-T4Circ-PostBglii-APSamples.png
Gel image-Amp/Phusion samples (G,H)