Daniel:Notebook/ComboLock/2017-4-29: Difference between revisions

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>Djacobse
(Created page with "=Production Run (Started Yesterday)= Back to Calendar ==Protocol-Part 4== <ol start="7"> <li>Exonuclea...")
 
>Djacobse
 
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=Production Run (Started [[Daniel:Notebook/ComboLock/2017-4-30|Yesterday]])=
=Production Run (Started [[Daniel:Notebook/ComboLock/2017-4-28|Yesterday]])=


[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]
Line 8: Line 8:
<li>Exonuclease Digestion</li>
<li>Exonuclease Digestion</li>
<ol type="A">
<ol type="A">
<li>Mix 25 uL Exo I (20U/uL) and 5 uL ExoIII (100U/uL)</li>
<li>Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)</li>
<li>Add 2 uL to each sample, mix by swirling pipette tip</li>
<li>Add 2 uL to each sample, mix by swirling pipette tip</li>
<li>Incubate for 1.5 hours at 37C</li>
<li>Incubate for 2 hours at 37C</li>
<li>Heat kill by incubating for 5 min at 95C</li>
<li>Heat kill by incubating for 5 min at 95C</li>
</ol>
</ol>
Line 37: Line 37:


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | RCA Primer (1 uM each)
| height="15"  valign="bottom" | p12RC*** (10 uM)
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 0.2
| align="center" align="center" valign="bottom" | 20.4
| align="center" align="center" valign="bottom" | 2.04
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 1.24


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| height="15"  valign="bottom" | RCA Primer LLRC*** (10 uM)
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 0.2
| align="center" align="center" valign="bottom" | 51
| align="center" align="center" valign="bottom" | 2.04
| align="center" align="center" valign="bottom" | 31
| align="center" align="center" valign="bottom" | 1.24


|- style="font-size:12pt"
|- 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" | 5.1
| align="center" align="center" valign="bottom" | 3.1
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer
| height="15"  valign="bottom" | 10X Buffer
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
Line 54: Line 60:
| align="center" align="center" valign="bottom" | 12.4
| align="center" align="center" valign="bottom" | 12.4


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| height="15"  valign="bottom" | Phi29
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 10.2
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 6.2
| align="center" align="center" valign="bottom" | 0


|- style="font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | BSA (10 mg/mL)
| height="15"  valign="bottom" | BSA (10 mg/mL)
| align="center" align="center" valign="bottom" | 0.4
| align="center" align="center" valign="bottom" | 0.4
Line 66: Line 72:
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 5.6
| align="center" align="center" valign="bottom" | 11.7
| align="center" align="center" valign="bottom" | 57.12
| align="center" align="center" valign="bottom" | 119.34
| align="center" align="center" valign="bottom" | 37.2
| align="center" align="center" valign="bottom" | 75.02


|- style="font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Total
| 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" | 20
|style="font-weight:bold" align="center" align="center" valign="bottom" | 163.2
|style="font-weight:bold" align="center" align="center" valign="bottom" | 153
|style="font-weight:bold" align="center" align="center" valign="bottom" | 99.2
|style="font-weight:bold" align="center" align="center" valign="bottom" | 93


|}
|}


<li>Add 15 uL master mix to each tube</li>
<li>Add 15 uL master mix to each tube</li>
<ol type="a"><li>Samples "A-C" get + master mixes; samples "D-F" get - master mixes</li></ol>
<li>Add 4 uL sample to appropriate tubes</li>
<li>Add 1 uM RCA primer and to new reaction tubes according to following rules</li>
<ol type="a">
<li>"A,D" samples get 2 uL each LLRC & primer12RC </li>
<li>"B,E" samples get 2 uL each LLRC*** & primer12RC </li>
<li>"C,F" samples get 4 uL LLRC***</li></ol>
<li>Heat reactions up to 95C for 5 min</li>
<li>Heat reactions up to 95C for 5 min</li>
<li>Cool to 55C and incubate for 15 minutes</li>
<li>Cool to 55C and incubate for 15 minutes</li>
<li>Cool to 30C and add enzyme</li>
<li>Cool to 30C and add 1 uL enzyme</li>
<li>Incubate for 3 hours at 30C</li>
<li>Incubate for 3 hours at 30C</li>
<li>Incubate for 65 minutes at 20C to denature</li>
<li>Incubate for 65 minutes at 20C to denature</li>
</ol></ol>
</ol>
<li>qPCR</li>
<li>Bglii digestion</li>
<ol type="A">
<ol type="A">
<li>Make qPCR master mixes according to following recipe</li>
<li>Make the following reactions; '''Do not add Bglii yet'''</li>
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt;font-weight:bold" align="center"
|style="background-color:#8DB4E2" width="145" height="30" | Reagent
|style="background-color:#8DB4E2" width="85" | Single Rxn Vol
|style="background-color:#95B3D7" width="85" | Master Mix (16.2X)
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | RCA Rxn
| align="center" align="center" valign="bottom" | 7
| align="center" align="center" valign="bottom" | 0
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer 3.1
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 32.4
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Bglii
| 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" | 10
| align="center" align="center" valign="bottom" | 162
 
|- 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" | 194.4
 
|}
 
<li>Aliquot 12 uL master mix into new tube</li>
<li>Add 7 uL appropriate sample</li>
<li>Mix with vortexer, spin down, and add 1 uL Bglii</li>
<li>Incubate 2 hr 37C</li>
<li>Heat kill with 65C for 20 min</li>
<li>Hold at 12C</ol>


Continued [[Daniel:Notebook/ComboLock/2017-5-1|Monday]]


[[Category:ComboLock]] [[Category:20170429]]
[[Category:ComboLock]] [[Category:20170428]]

Latest revision as of 18:54, 1 May 2017

Production Run (Started Yesterday)[edit]

Back to Calendar

Protocol-Part 4[edit]

  1. Exonuclease Digestion
    1. Mix 17.5 uL Exo I (20U/uL) and 3.5 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
  2. Rolling Circle Amplification
    1. Prepare master mix according to table below
    2. Reagent Single Rxn (uL) Master Mix X (10.2X) Master Mix Y (6.2X)
      Enzyme NA NEB Thermo
      Sample 4 0 0
      p12RC*** (10 uM) 0.2 2.04 1.24
      RCA Primer LLRC*** (10 uM) 0.2 2.04 1.24
      dNTP (10 mM) 0.5 5.1 3.1
      10X Buffer 2 20.4 12.4
      Phi29 1 0 0
      BSA (10 mg/mL) 0.4 4.08 0
      nfH2O 11.7 119.34 75.02
      Total 20 153 93
    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 65 minutes at 20C to denature
  3. Bglii digestion
    1. Make the following reactions; Do not add Bglii yet
    2. Reagent Single Rxn Vol Master Mix (16.2X)
      RCA Rxn 7 0
      10X Buffer 3.1 2 32.4
      Bglii 1 0
      nfH2O 10 162
      Total 20 194.4
    3. Aliquot 12 uL master mix into new tube
    4. Add 7 uL appropriate sample
    5. Mix with vortexer, spin down, and add 1 uL Bglii
    6. Incubate 2 hr 37C
    7. Heat kill with 65C for 20 min
    8. Hold at 12C

    Continued Monday