Daniel:Notebook/ComboLock/2016-11-30: Difference between revisions

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>Djacobse
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<li>Incubate at 37C for 3 hours</li>
<li>Incubate at 37C for 3 hours</li>
<li>Heat kill by incubating at 65C for 10 minutes</li></ol>
<li>Heat kill by incubating at 65C for 10 minutes</li></ol>
<li>qPCR</li>
<ol type="A">
{| 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="100" height="30" | Sample
| width="120" | Condition
| width="85" | Lane
| width="85" | AmpR Index
| width="85" | Sample Vol (uL)
| width="100" | 2X Kapa SYBR qPCR MM
| width="100" | 10 uM Forward Primer
| width="100" | 10 uM Reverse Primer
| width="85" | H2O
| width="90" | 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" | 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" | 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" | 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" | 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 Phosphate
| 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 Phosphate
| 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 C Probe
| 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 C Probe
| 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"  valign="bottom" | NTC
| align="center" valign="bottom" | qPCR Primers Only
| 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" | 23
| align="center" align="center" | 50
|}
</ol>
</ol>

Revision as of 16:34, 30 November 2016

Extended Latch Test (Started [[Daniel:Notebook/ComboLock/2016-11-29])

Back to Calendar

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C Probe
Sample 4 (AB) No Kinase

Protocol

  1. Exonuclease Digestion
    1. Combine 15 uL of Exonuclease I (20U/uL) and 3 uL Exonuclease III (100U/uL)
    2. Add 2 uL of mix to each reaction
    3. Incubate for 30 minutes to 2 hours at 37C
    4. Heat kill by incubating for 5 minutes at 94C
  2. Rolling Circle Amplification
    1. Prepare 9X master mix according to table below
    2. Reagent Stock Concentration Final Conc/Amt uL Added in 20 uL Rxn Master Mix (9X)
      Template NA NA 5 45
      RCA Primer 10 uM 2 uM 4 36
      dNTP 1 mM 50 uM 1 9
      10X Buffer 10X 1X 2 18
      Phi29 (NEB) 10 U/uL 10 U 1 9
      nfH2O NA NA 7 63
      Total     20 180
    3. Add 15 uL master mix to each tube
    4. Add 5 uL sample to correct reaction tubes
    5. Incubate at 37C for 3 hours
    6. Heat kill by incubating at 65C for 10 minutes
  3. qPCR
    1. 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 20 2 25 1 1 21 50
      Sample 1B Sample A2 20 2 25 1 1 21 50
      Sample 2A No Template A3 21 2 25 1 1 21 50
      Sample 2B No Template A4 21 2 25 1 1 21 50
      Sample 3A No Phosphate A5 22 2 25 1 1 21 50
      Sample 3B No Phosphate A6 22 2 25 1 1 21 50
      Sample 4A No C Probe A7 23 2 25 1 1 21 50
      Sample 4B No C Probe A8 23 2 25 1 1 21 50
      NTC qPCR Primers Only H1 24 2 25 1 1 23 50