Daniel:Notebook/ComboLock/2017-2-8: Difference between revisions

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>Djacobse
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=Title=
=v3 Oligos-Round 3=


[[Daniel:Notebook/ComboLock|Back to Calendar]]
==Protocol==
<ol start="9">
<li>qPCR</li>
<ol type="A">
<li>Make 21.2X qPCR master mix according to following recipe</li>
<ol type="a">
<li>445.2 uL nfH2O</li>
<li>530 uL SYBR Fast</li>
<li>21.2 uL 10 uM AmpF</li>
</ol>
<li>Add 47 uL master mix to each well</li>
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table</li>


[[Daniel:Notebook/ComboLock|Back to Calendar]]
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center"
| width="110" height="45" | Sample
| width="180" | Condition
| width="65" | Pre/Post RCA
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | Sample Vol (uL)
| width="95" | 2X Kapa SYBR qPCR MM
| width="95" | 10 uM Forward Primer
| width="95" | 10 uM Reverse Primer
| width="65" | H2O
| width="65" | Total Volume (uL)
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1A
| align="center" | Sample
| align="center" | Pre
| align="center" | A1
| align="center" align="center" | 20
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1B
| align="center" | Sample
| align="center" | Pre
| align="center" | A2
| align="center" align="center" | 20
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 2A
| align="center" | No Template
| align="center" | Pre
| align="center" | A3
| align="center" align="center" | 21
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 2B
| align="center" valign="bottom" | No Template
| align="center" valign="bottom" | Pre
| align="center" | A4
| align="center" align="center" | 21
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 3A
| align="center" | No C Probe
| align="center" | Pre
| align="center" | A5
| align="center" align="center" | 22
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 3B
| align="center" | No C Probe
| align="center" | Pre
| align="center" | A6
| align="center" align="center" | 22
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 4A
| align="center" | No Lock Phosphate
| align="center" | Pre
| align="center" | A7
| align="center" align="center" | 23
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 4B
| align="center" | No Lock Phosphate
| align="center" valign="bottom" | Pre
| align="center" | A8
| align="center" align="center" | 23
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1AX
| align="center" | Sample
| align="center" | Post
| align="center" | H1
| align="center" align="center" | 24
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1BX
| align="center" | Sample
| align="center" | Post
| align="center" | H2
| align="center" align="center" | 24
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 2AX
| align="center" | No Template
| align="center" | Post
| align="center" | H3
| align="center" align="center" | 25
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Sample 2BX
| align="center" valign="bottom" | No Template
| align="center" valign="bottom" | Post
| align="center" | H4
| align="center" align="center" | 25
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 3AX
| align="center" | No C Probe
| align="center" | Post
| align="center" | H5
| align="center" align="center" | 26
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 3BX
| align="center" | No C Probe
| align="center" | Post
| align="center" | H6
| align="center" align="center" | 26
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 4AX
| align="center" | No Lock Phosphate
| align="center" | Post
| align="center" | H7
| align="center" align="center" | 27
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="background-color:#DA9694;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 4BX
| align="center" | No Lock Phosphate
| align="center" valign="bottom" | Post
| align="center" | H8
| align="center" align="center" | 27
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | NTC
| align="center" valign="bottom" | qPCR Primers Only
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" | D5
| align="center" align="center" | 30
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 23
| align="center" align="center" | 50
 
|}
 
<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 x24</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol></ol>
<li>TBE Gel</li>
<ol type="A">
<li>Mix 224 uL TBE, 56 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===


<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=2 heights=300px widths=300px>

Revision as of 20:29, 8 February 2017

v3 Oligos-Round 3

Back to Calendar

Protocol

  1. qPCR
    1. Make 21.2X qPCR master mix according to following recipe
      1. 445.2 uL nfH2O
      2. 530 uL SYBR Fast
      3. 21.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
    4. Sample Condition Pre/Post 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 Sample Pre A1 20 2 25 1 1 21 50
      Sample 1B Sample Pre A2 20 2 25 1 1 21 50
      Sample 2A No Template Pre A3 21 2 25 1 1 21 50
      Sample 2B No Template Pre A4 21 2 25 1 1 21 50
      Sample 3A No C Probe Pre A5 22 2 25 1 1 21 50
      Sample 3B No C Probe Pre A6 22 2 25 1 1 21 50
      Sample 4A No Lock Phosphate Pre A7 23 2 25 1 1 21 50
      Sample 4B No Lock Phosphate Pre A8 23 2 25 1 1 21 50
      Sample 1AX Sample Post H1 24 2 25 1 1 21 50
      Sample 1BX Sample Post H2 24 2 25 1 1 21 50
      Sample 2AX No Template Post H3 25 2 25 1 1 21 50
      Sample 2BX No Template Post H4 25 2 25 1 1 21 50
      Sample 3AX No C Probe Post H5 26 2 25 1 1 21 50
      Sample 3BX No C Probe Post H6 26 2 25 1 1 21 50
      Sample 4AX No Lock Phosphate Post H7 27 2 25 1 1 21 50
      Sample 4BX No Lock Phosphate Post H8 27 2 25 1 1 21 50
      NTC qPCR Primers Only   D5 30 2 25 1 1 23 50
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x24
      7. 72C 2 min
      8. 16C hold
  2. TBE Gel
    1. Mix 224 uL TBE, 56 uL 6x loading dye
    2. Aliquot 10 uL per sample/ladder lane onto parafilm
    3. Add 2 uL of sample or ladder to correct drop
    4. Load 10 uL in to well
    5. Run gel for 23 minutes at 230V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc


Results