Daniel:Notebook/ComboLock/2017-1-14: Difference between revisions

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>Djacobse
 
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<li>Heat kill by incubating for 5 min at 95C</li>
<li>Heat kill by incubating for 5 min at 95C</li>
</ol>
</ol>
******
<li>Rolling Circle Amplification</li>
<li>Rolling Circle Amplification</li>
<ol type="A">
<ol type="A">
Line 19: Line 20:
<li>Add sample to new reaction tubes according to following table</li>
<li>Add sample to new reaction tubes according to following table</li>


{| 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="160" height="30" | Reagent
| width="65" | uL Added
| width="90" | Master Mix (8.5X)
|- style="font-size:12pt"
| height="15"  valign="bottom" | Template
| align="center" align="center" valign="bottom" | 5
| align="center" valign="bottom" | NA
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | RCA Primer (10 uM)
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 21.25
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| align="center" align="center" valign="bottom" | 0.8
| align="center" align="center" valign="bottom" | 6.8
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 17
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 8.5
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 8.7
| align="center" align="center" valign="bottom" | 73.95
|- 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" | 127.5
|}


<li>Incubate at 37C for 3 hours</li>
<li>Incubate at 37C for 3 hours</li>
Line 34: Line 77:
</ol>
</ol>
<li>Add 47 uL master mix to each well</li>
<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 in the following section</li>
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table</li>
 
{| 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="130" height="45" | Sample
| width="100" | Condition
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | Sample Vol (uL)
| width="78" | 2X Kapa SYBR qPCR MM
| width="62" | 10 uM Forward Primer
| width="65" | 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" | A1
| 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"  | Sample 1B
| align="center" | Sample
| align="center" | A2
| 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 2A
| align="center" | No Template
| align="center" | A3
| 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"  valign="bottom" | Sample 2B
| align="center" valign="bottom" | No Template
| align="center" | A4
| 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 3A
| align="center" | No C Probe
| align="center" | A5
| 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 3B
| align="center" | No C Probe
| align="center" | A6
| 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="30"  | Sample 4A
| align="center" | No Lock Phosphate
| align="center" | A7
| 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:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="30"  | Sample 4B
| align="center" | No Lock Phosphate
| align="center" | A8
| 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="30"  valign="bottom" | NTC
| align="center" valign="bottom" | qPCR Primers Only
| align="center" | H1
| 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" | 23
| align="center" align="center" | 50
 
|}


<li>qPCR Cycles</li>
<li>qPCR Cycles</li>
Line 61: Line 227:


<gallery perrow=2 heights=300px widths=300px>
<gallery perrow=2 heights=300px widths=300px>
File:|qPCR Curve
File:20170114-qPCR-v3Oligos.png|qPCR Curve
File:|Gel Image
File:2017-01-14-v3Oligos.png|Gel Image
</gallery>
</gallery>




[[Category:ComboLock]] [[Category:20170113]]
[[Category:ComboLock]] [[Category:20170113]]

Latest revision as of 01:38, 15 January 2017

Version 3 Oligos (Started Yesterday)[edit]

Back to Calendar

Protocol-Part3[edit]

  1. Exonuclease Digestion
    1. Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  2. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table
    4. Reagent uL Added Master Mix (8.5X)
      Template 5 NA
      RCA Primer (10 uM) 2.5 21.25
      dNTP (1 mM) 0.8 6.8
      10X Buffer 2 17
      Phi29 1 8.5
      nfH2O 8.7 73.95
      Total 20 127.5
    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step

    Protocol-Part4[edit]

  3. qPCR
    1. Make 9.2X qPCR master mix according to following recipe
      1. 193.2 uL nfH2O
      2. 230 uL SYBR Fast
      3. 9.2 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 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 A1 21 2 25 1 1 21 50
      Sample 1B Sample A2 21 2 25 1 1 21 50
      Sample 2A No Template A3 22 2 25 1 1 21 50
      Sample 2B No Template A4 22 2 25 1 1 21 50
      Sample 3A No C Probe A5 23 2 25 1 1 21 50
      Sample 3B No C Probe A6 23 2 25 1 1 21 50
      Sample 4A No Lock Phosphate A7 24 2 25 1 1 21 50
      Sample 4B No Lock Phosphate A8 24 2 25 1 1 21 50
      NTC qPCR Primers Only H1 25 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
  4. TBE Gel
    1. Mix 80 uL TBE, 20 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 22 minutes at 250V
    6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
    7. Rinse gel and image in gel doc

Results[edit]