Daniel:Notebook/ComboLock/2017-6-15: Difference between revisions

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=Antibody Detection Test-Positive Control (Started [[Daniel:Notebook/ComboLock/2017-6-14|Yesterday]])=
=Antibody Detection Test-Positive Control (Started [[Daniel:Notebook/ComboLock/2017-6-14|Yesterday]])=


[[Daniel:Notebook/ComboLock|Back to Calendar]]=
[[Daniel:Notebook/ComboLock|Back to Calendar]]


==Protocol-Part 3==
==Protocol-Part 3==
Line 41: Line 41:
| align="center" | 40 nmol
| align="center" | 40 nmol
| align="center" align="center" | 8
| align="center" align="center" | 8
| align="center" align="center" | 84
| align="center" align="center" | 52


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
Line 48: Line 48:
| align="center" | 600 pmol
| align="center" | 600 pmol
| align="center" align="center" | 0.6
| align="center" align="center" | 0.6
| align="center" align="center" | 6.3
| align="center" align="center" | 3.9


|- style="font-size:12pt"
|- style="font-size:12pt"
Line 55: Line 55:
| align="center" | 15 umol
| align="center" | 15 umol
| align="center" align="center" | 3
| align="center" align="center" | 3
| align="center" align="center" | 31.5
| align="center" align="center" | 19.5


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
Line 62: Line 62:
| align="center" | 1X
| align="center" | 1X
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" | 21
| align="center" align="center" | 13


|- style="font-size:12pt"
|- style="font-size:12pt"
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| align="center" | 10 U
| align="center" | 10 U
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" | 21
| align="center" align="center" | 13


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="30"  valign="bottom" | Phusion HF DNA Polymerase
| height="15"  valign="bottom" | Phusion HF DNA Polymerase
| align="center" | 2000 U/mL
| align="center" | 2000 U/mL
| align="center" | 6.4U
| align="center" | 6.4U
| align="center" align="center" | 3.2
| align="center" align="center" | 3.2
| align="center" align="center" | 33.6
| align="center" align="center" | 20.8


|- style="font-size:12pt"
|- style="font-size:12pt"
Line 83: Line 83:
| align="center" align="center" |  
| align="center" align="center" |  
| align="center" align="center" | 1.2
| align="center" align="center" | 1.2
| align="center" align="center" | 12.6
| align="center" align="center" | 7.8


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
Line 90: Line 90:
| align="center" align="center" |  
| align="center" align="center" |  
|style="font-weight:bold" align="center" align="center" | 20
|style="font-weight:bold" align="center" align="center" | 20
|style="font-weight:bold" align="center" align="center" | 210
|style="font-weight:bold" align="center" align="center" | 130


|}
|}
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<li>Incubate at 95C for 5 minutes</li>
<li>Incubate at 95C for 5 minutes</li>
<li>Hold at 16C</li>
<li>Hold at 16C</li>
</ol></ol>
</ol>
<li>Exonuclease Digestion</li>
<ol type="A">
<li>Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)</li>
<li>Add 2 uL to each sample, mix by swirling pipette tip</li>
<li>Incubate for 2 hours at 37C</li>
<li>Heat kill by incubating for 5 min at 95C</li>
</ol>
</ol>
 
==Protocol-Part 4==
 
<ol start="7">
<li>Rolling Circle Amplification</li>
<ol type="A">
<li>Prepare master mix according to table below</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="190" height="30" | Reagent
| width="95" | Single Rxn (uL)
| width="100" | Master Mix X (6.2X)
 
|- style="font-size:12pt"
| height="15"  | Enzyme
| align="center" | NA
| align="center" | Thermo
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 0
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | p12RC*** (10 uM)
| align="center" align="center" valign="bottom" | 0.2
| align="center" align="center" valign="bottom" | 1.24
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | RCA Primer LLRC*** (10 uM)
| align="center" align="center" valign="bottom" | 0.2
| align="center" align="center" valign="bottom" | 1.24
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (10 mM)
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 3.1
 
|- 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" | 12.4
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 0
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 11.7
| align="center" align="center" valign="bottom" | 72.54
 
|- 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" | 90.52
 
|}
 
<li>Add 15 uL master mix to each tube</li>
<li>Add 4 uL sample to appropriate tubes</li>
<li>Heat reactions up to 95C for 5 min</li>
<li>Cool to 55C and incubate for 15 minutes</li>
<li>Cool to 30C and add 1 uL enzyme</li>
<li>Incubate for 3 hours at 30C</li>
<li>Incubate for 20 minutes at 65C to denature</li>
</ol>
 
 
[[Category:ComboLock]] [[Category:20170614]]

Latest revision as of 18:55, 16 June 2017

Antibody Detection Test-Positive Control (Started Yesterday)[edit]

Back to Calendar

Protocol-Part 3[edit]

  1. Latch and Bolt Hybridization/Ligation
    1. Heat kill enzyme by incubating at 65C for 10 minutes
    ******
    
  2. Padlock Hybridization
    1. Add 20 uL of following 6.2X Master Mix
      1. 6.2 uL 10 uM Padlock0601
      2. 12.4 uL 10X Amp Ligase buffer
      3. 105.4 uL nfH2O
    2. Incubate at 70C for 5 min
    3. Incubate at 50C for 15 minutes
    4. Wash once with wash buffer*Do not wash sample 5
    5. Resuspend in 20 uL 1X Amp Ligase buffer
    ******
    
  3. Circularization
    1. For phusion samples, make the following master mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (10.5x) (uL)
      NAD+ 5 mM 40 nmol 8 52
      dNTP 1 mM 600 pmol 0.6 3.9
      Betaine 5 M 15 umol 3 19.5
      10X AmpLigase Buffer 10X 1X 2 13
      Amp Ligase 5 U/uL 10 U 2 13
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 20.8
      nf H2O     1.2 7.8
      Total     20 130
    3. Add 20 uL circularization mix to each sample
    4. Incubate at 55C for 2 hours
    5. Incubate at 95C for 5 minutes
    6. Hold at 16C
  4. Exonuclease Digestion
    1. Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 2 hours at 37C
    4. Heat kill by incubating for 5 min at 95C

Protocol-Part 4[edit]

  1. Rolling Circle Amplification
    1. Prepare master mix according to table below
    2. Reagent Single Rxn (uL) Master Mix X (6.2X)
      Enzyme NA Thermo
      Sample 4 0
      p12RC*** (10 uM) 0.2 1.24
      RCA Primer LLRC*** (10 uM) 0.2 1.24
      dNTP (10 mM) 0.5 3.1
      10X Buffer 2 12.4
      Phi29 1 0
      nfH2O 11.7 72.54
      Total 20 90.52
    3. Add 15 uL master mix to each tube
    4. Add 4 uL sample to appropriate tubes
    5. Heat reactions up to 95C for 5 min
    6. Cool to 55C and incubate for 15 minutes
    7. Cool to 30C and add 1 uL enzyme
    8. Incubate for 3 hours at 30C
    9. Incubate for 20 minutes at 65C to denature