Daniel:Notebook/ComboLock/2017-4-5: Difference between revisions
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>Djacobse (Created page with "=RCA Test (Started March 24)= Back to Calendar ==Second Strand Synthesis part II== Since the second st...") |
>Djacobse |
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(10 intermediate revisions by the same user not shown) | |||
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<li>Goto (b) x20</li> | <li>Goto (b) x20</li> | ||
<li>72C for 2 min</li></ol> | <li>72C for 2 min</li></ol> | ||
</ol></ol> | </ol> | ||
<li>Bgl Digestion</li> | |||
<ol type="A"> | |||
<li>Make the following reactions; '''Do not add Bglii yet'''</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt;font-weight:bold" align="center" | |||
|style="background-color:#8DB4E2" width="180" height="30" | Reagent | |||
|style="background-color:#8DB4E2" width="85" | Single Rxn Vol | |||
|style="background-color:#95B3D7" width="85" | Master Mix (12.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RCA Rxn | |||
| align="center" align="center" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer 3.1 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 24.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Bglii | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 9 | |||
| align="center" align="center" valign="bottom" | 111.6 | |||
|- style="font-size:12pt" | |||
| 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" | 136 | |||
|} | |||
<li>Aliquot 11 uL master mix into each sample</li> | |||
<li>Add appropriate sample and 1 uL Bglii to each</li> | |||
<li>Incubate 1 hr 37C</li> | |||
<li>Heat kill with 65C for 20 min</li> | |||
</ol> | |||
<li>TBE Gel-PreBgl</li> | |||
<ol type="A"> | |||
<li>Mix 96 uL 10X TBE and 24 uL 6X dye</li> | |||
<li>Aliquot 10 uL on to parafilm per sample</li> | |||
<li>Add 2 uL reaction or 1.5 uL ladder to appropriate aliquot</li> | |||
<li>Add 10 uL mix to gel lanes</li> | |||
[[Image:GelLanes-20170405-RCATest-CPLP-PostqPCR-PreBgl.png|400px]] | |||
<li>Run gel for 24 minutes at 230V</li> | |||
<li>Stain with 2 uL SYBR gold for 3 minutes</li> | |||
<li>Rinse and image in gel doc</li> | |||
</ol> | |||
<li>TBE Gel-PostBgl</li> | |||
<ol type="A"> | |||
<li>Mix 120 uL 10X TBE and 40 uL 6X dye</li> | |||
<li>Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot</li> | |||
<li>Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot</li> | |||
<li>Add 10 uL mix to gel lanes</li> | |||
<li>Run gel for 24 minutes at 230V</li> | |||
<li>Stain with 2 uL SYBR gold for 3 minutes</li> | |||
<li>Rinse and image in gel doc</li> | |||
</ol> | |||
</ol> | |||
===Results=== | |||
<gallery perrow=3 heights=300px widths=300px> | |||
File:20170405-qPCR-RCATest-CPLP-ssSynthesis2.png|qPCR curves | |||
File:2017-04-05_-RCATest-CPLP-ssSynthesis2-PreBgl.png|Gel image-pre Bgl digestion | |||
File:2017-04-05_-RCATest-CPLP-ssSynthesis2-PostBgl.png|Gel image-post Bgl digestion | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20170324]] |
Latest revision as of 15:34, 7 April 2017
RCA Test (Started March 24)[edit]
Second Strand Synthesis part II[edit]
Since the second strand synthesis from Monday worked well, I'm going to repeat the experiment but using the NEB 3 hr and 6 hr samples (X and P, respectively). I won't bother with the Bgl digested ones as I'll just digest after.
- qPCR-Second strand synthesis
- Make the following 14.2X master mix
- 14.2 uL AmpF-6.4Sol (10 uM)
- 284 uL Kapa SYBR 2X Master Mix
- 241.4 uL nfH2O
- Aliquot 38 uL master mix into each lane
- Add 2 uL sample according to plate layout File:PlateLayout-qPCR-20170405-RCATest-CPLP-ssSynthesis2.png
- Run the following protocol
- 95C for 2 min
- 95C for 5 sec
- 55C for 30 sec
- 72C for 45 sec
- Goto (b) x20
- 72C for 2 min
- Bgl Digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 11 uL master mix into each sample
- Add appropriate sample and 1 uL Bglii to each
- Incubate 1 hr 37C
- Heat kill with 65C for 20 min
- TBE Gel-PreBgl
- Mix 96 uL 10X TBE and 24 uL 6X dye
- Aliquot 10 uL on to parafilm per sample
- Add 2 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes File:GelLanes-20170405-RCATest-CPLP-PostqPCR-PreBgl.png
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
- TBE Gel-PostBgl
- Mix 120 uL 10X TBE and 40 uL 6X dye
- Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot
- Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
Reagent | Single Rxn Vol | Master Mix (12.2X) |
RCA Rxn | 8 | 0 |
10X Buffer 3.1 | 2 | 24.4 |
Bglii | 1 | 0 |
nfH2O | 9 | 111.6 |
Total | 20 | 136 |
Results[edit]
- 20170405-qPCR-RCATest-CPLP-ssSynthesis2.png
qPCR curves
- 2017-04-05 -RCATest-CPLP-ssSynthesis2-PreBgl.png
Gel image-pre Bgl digestion
- 2017-04-05 -RCATest-CPLP-ssSynthesis2-PostBgl.png
Gel image-post Bgl digestion