Daniel:Notebook/PosSequencing/2017-6-13: Difference between revisions

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>Djacobse
(Created page with "=Biotin vs Dual Biotin= Back to Calendar '''Workflow Reminder''' 350x265px ==Prot...")
 
>Djacobse
 
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=Biotin vs Dual Biotin=
=Biotin vs Dual Biotin (Started [[Daniel:Notebook/PosSequencing/2017-6-12|Yesterday]])=


[[Daniel:Notebook/PosSequencing|Back to Calendar]]
[[Daniel:Notebook/PosSequencing|Back to Calendar]]
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<li>Prepare 2 plates; 1 single biotin and 1 dual biotin</li>
<li>Prepare 2 plates; 1 single biotin and 1 dual biotin</li>
<li>For each sample prepare 58X master mix and (4X) master mix: 1X components set to 30 uL; add 2uL sample</li>
<li>For each sample prepare 58X master mix and (4X) master mix: 1X components set to 30 uL; add 2uL sample</li>
<li>ol type="a">
<ol type="a">
<li>58 (4) uL 1 uM primer2</li>
<li>58 (4) uL 1 uM primer2</li>
<li>(4) uL 1 uM biotin/2biotin uracil primer</li>
<li>(4) uL 1 uM biotin/2biotin uracil primer</li>
Line 21: Line 21:
<li>870 (60) uL 2X Kapa SYBR</li>
<li>870 (60) uL 2X Kapa SYBR</li>
<li>638 (44) uL nfH<sub>2</sub>O</li></ol>
<li>638 (44) uL nfH<sub>2</sub>O</li></ol>
<li>Add 28 uL master mix according to plate (4X master mix is for positive controls, 58X is for samples and negative controls)</li>
<li>Add 28 uL master mix according to plate layout (4X master mix is for positive controls, 58X is for samples and negative controls)</li>
 
[[Image:PlateLayout-20170613-BiotinTest.png|350x250px]]
 
<li>Add 2 uL appropriate sample according to plate layout</li>
<li>Add 2 uL appropriate sample according to plate layout</li>
<li>Use "standard" thermocycling program</li>
<li>Use "standard" thermocycling program</li>
</ol>
</ol>
==Results==
<gallery perrow=2 heights=250px widths=350px mode=packed-hover caption="Single Biotin Results">
File:PlateLayout-20170613-BiotinTest.png|Plate array
File:Singlebiotin-20170613-plateCTs.png|Plate CTs
File:Singlebiotin-20170613-plateCTs-normed.png|Normalized plate CTs
File:Singlebiotin-20170613-rawcurves.png|Raw curves
</gallery>
<gallery perrow=2 heights=250px widths=350px mode=packed-hover caption="Single Biotin Results">
File:PlateLayout-20170613-BiotinTest.png|Plate array
File:Dualbiotin-20170613-plateCTs.png|Plate CTs
File:Dualbiotin-20170613-plateCTs-normed.png|Normalized plate CTs
File:Dualbiotin-20170613-rawcurves.png|Raw curves
</gallery>
==Discussion==
I won't bother to do any more complicated analysis than what's above. If you look at the plate CTs the pattern emerges that the dual biotin is far more resilient than the single biotin. At 95C it does experience some losses, but 90C appears to hold well. I can probably get away with short 95C or 90C incubations for doing emPCR.
[[Category:PosSeq]] [[Category:20170612]]

Latest revision as of 17:06, 14 June 2017

Biotin vs Dual Biotin (Started Yesterday)[edit]

Back to Calendar


Workflow Reminder

File:ExperimentMap-20170610-Biotin-DualBiotin.png

Protocol[edit]

  1. qPCR
    1. Prepare 2 plates; 1 single biotin and 1 dual biotin
    2. For each sample prepare 58X master mix and (4X) master mix: 1X components set to 30 uL; add 2uL sample
      1. 58 (4) uL 1 uM primer2
      2. (4) uL 1 uM biotin/2biotin uracil primer
      3. 58 uL 10 pM barcode oligo v2
      4. 870 (60) uL 2X Kapa SYBR
      5. 638 (44) uL nfH2O
    3. Add 28 uL master mix according to plate layout (4X master mix is for positive controls, 58X is for samples and negative controls)
    4. File:PlateLayout-20170613-BiotinTest.png
    5. Add 2 uL appropriate sample according to plate layout
    6. Use "standard" thermocycling program

    Results[edit]

    Discussion[edit]

    I won't bother to do any more complicated analysis than what's above. If you look at the plate CTs the pattern emerges that the dual biotin is far more resilient than the single biotin. At 95C it does experience some losses, but 90C appears to hold well. I can probably get away with short 95C or 90C incubations for doing emPCR.