Daniel:Notebook/ComboLock/2017-3-3: Difference between revisions
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>Djacobse (Created page with "=Padlock0601 Test (Started Yesterday)= Back to Calendar ==Protocol-Part 5== ===Results=== <gallery per...") |
>Djacobse |
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=Padlock0601 Test (Started [[Daniel:Notebook/ComboLock/2017-3-1| | =Padlock0601 Test (Started [[Daniel:Notebook/ComboLock/2017-3-1|Wednesday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
==Protocol-Part 5== | ==Protocol-Part 5== | ||
<ol start="11"> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 240 uL TBE, 60 uL 6x loading dye</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>Load 10 uL in to well</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>Rinse gel and image in gel doc</li> | |||
</ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make 9.2X qPCR master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>193.2 uL nfH2O</li> | |||
<li>230 uL SYBR Fast</li> | |||
<li>9.2 uL 10 uM AmpF</li> | |||
<li>9.2 uL 10 uM AmpR-Ind20</li> | |||
</ol> | |||
<li>Add 48 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to plate layout</li> | |||
[[Image:PlateLayout-20170303-Padlock0601-Bglii.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 96 uL TBE, 24 uL 6x loading dye</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>Load 10 uL in to well</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>Rinse gel and image in gel doc</li> | |||
</ol></ol> | |||
===Results=== | ===Results=== | ||
<gallery perrow= | <gallery perrow=3 heights=300px widths=300px caption="qPCR curves and gel images for samples. See individual gel captions for more info. Note that for all gel images Post-RCA the left sample was RC amplified with normal primer and the right sample with phosphothiorate primer"> | ||
File:20170302-qPCR-Padlock0601Test.png|qPCR curves for run on 3-2-2017 | |||
File:2017-03-03-Padlock0601-PreRCA-Unpurifed.png|Gel image-PreRCA Unpurified | File:2017-03-03-Padlock0601-PreRCA-Unpurifed.png|Gel image-PreRCA Unpurified | ||
File:2017-03-03-Padlock0601-PreRCA-Purifed.png|Gel image-PreRCA Purifed | File:2017-03-03-Padlock0601-PreRCA-Purifed.png|Gel image-PreRCA Purifed | ||
File:2017-03-03-Padlock0601-PostRCA-Unpurifed.png|Gel image-PostRCA Unpurified | File:2017-03-03-Padlock0601-PostRCA-Unpurifed.png|Gel image-PostRCA Unpurified | ||
File:2017-03-03-Padlock0601-PostRCA-Purifed.png|Gel image-PostRCA Purified | File:2017-03-03-Padlock0601-PostRCA-Purifed.png|Gel image-PostRCA Purified | ||
File:20170303-qPCR-Padlock0601-Bglii.png|qPCR curves for post-Bglii digestion | |||
File:2017-03-03-Padlock0601-Bglii.png|Post BglII digestion | File:2017-03-03-Padlock0601-Bglii.png|Post BglII digestion | ||
</gallery> | </gallery> |
Latest revision as of 23:03, 6 March 2017
Padlock0601 Test (Started Wednesday)[edit]
Protocol-Part 5[edit]
- 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- qPCR
- Make 9.2X qPCR master mix according to following recipe
- 193.2 uL nfH2O
- 230 uL SYBR Fast
- 9.2 uL 10 uM AmpF
- 9.2 uL 10 uM AmpR-Ind20
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170303-Padlock0601-Bglii.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 96 uL TBE, 24 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Results[edit]
- qPCR curves and gel images for samples. See individual gel captions for more info. Note that for all gel images Post-RCA the left sample was RC amplified with normal primer and the right sample with phosphothiorate primer
- 20170302-qPCR-Padlock0601Test.png
qPCR curves for run on 3-2-2017
- 2017-03-03-Padlock0601-PreRCA-Unpurifed.png
Gel image-PreRCA Unpurified
- 2017-03-03-Padlock0601-PreRCA-Purifed.png
Gel image-PreRCA Purifed
- 2017-03-03-Padlock0601-PostRCA-Unpurifed.png
Gel image-PostRCA Unpurified
- 2017-03-03-Padlock0601-PostRCA-Purifed.png
Gel image-PostRCA Purified
- 20170303-qPCR-Padlock0601-Bglii.png
qPCR curves for post-Bglii digestion
- 2017-03-03-Padlock0601-Bglii.png
Post BglII digestion