Daniel:Notebook/ComboLock/2017-1-27: Difference between revisions

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<li>Add 20 uL phusion mix to 20 uL reaction</li>
<li>Add 20 uL phusion mix to 20 uL reaction</li>
<li>Incubate at 55C for 4 hours</li>
<li>Incubate at 55C for 2 hours</li>
</ol></ol>
 
==Protocol-Part3==
 
<ol start="6">
<li>Exonuclease Digestion</li>
<ol type="A">
<li>Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)</li>
<li>Add 2 uL to each sample, mix by swirling pipette tip</li>
<li>Incubate for 1 hour at 37C</li>
<li>Heat kill by incubating for 5 min at 95C</li>
</ol>
</ol>
<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="180" height="30" | Reagent
| width="85" | uL Added
| width="90" | Master Mix (16.2X)
|- style="font-size:12pt"
| height="15"  valign="bottom" | Template
| align="center" align="center" valign="bottom" | 4
| 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" | 40.5
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 32.4
|- 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" | 32.4
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| align="center" align="center" valign="bottom" | 1
| align="center" valign="bottom" | NA
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | BSA (10 mg/mL)
| align="center" align="center" valign="bottom" | 0.4
| align="center" align="center" valign="bottom" | 6.48
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 8.1
| align="center" align="center" valign="bottom" | 131.22
|- style="background-color:#BFBFBF;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" | 243
|}
<li>Add 14 uL master mix to each tube</li>
<li>Add sample and 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:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
| width="55" height="33" | Sample Code
| width="85" | Phi29 Source
| width="85" | Incubation Time (hrs)
| width="80" | Sample (1AB)
| width="100" | No Template (2AB)
| width="110" | No C Probes (3AB)
| width="60" | No PO4 (4AB)
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | X
| NEB
| 3 hrs
|style="font-weight:bold" | X
|style="font-weight:bold" | X
|style="font-weight:bold" align="center" | &nbsp;
|style="font-weight:bold" align="center" | &nbsp;
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| height="15" | Y
| NEB
| 6 hrs
|style="font-weight:bold" | X
|style="font-weight:bold" align="center" | &nbsp;
|style="font-weight:bold" | X
|style="font-weight:bold" align="center" | &nbsp;
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | Z
| NEB
| 12 hrs
|style="font-weight:bold" | X
|style="font-weight:bold" align="center" | &nbsp;
|style="font-weight:bold" align="center" | &nbsp;
|style="font-weight:bold" | X
|- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom"
| height="15" | Q
| Epicentre
| 3 hrs
|style="font-weight:bold" | X
|style="font-weight:bold" align="center" | &nbsp;
|style="font-weight:bold" align="center" | &nbsp;
|style="font-weight:bold" | X
|}
<li>Incubate at 30C for X hours (see table above)</li>
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol>


Continued [[Daniel:Notebook/ComboLock/2017-1-28|tomorrow]]
Continued [[Daniel:Notebook/ComboLock/2017-1-28|tomorrow]]


[[Category:ComboLock]] [[Category:20170127]]
[[Category:ComboLock]] [[Category:20170127]]

Latest revision as of 23:46, 27 January 2017

RCA Timing Test (v3 Oligos)[edit]

Back to Calendar

I'm going to test the v3 oligos again, this time using longer RCA times than the other ones. This is from what I saw in the last experiment, which suggests to me that increased RCA time can potentially yield results. As such, I'll try several times to test the theory.

Protocol[edit]

  1. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine following into a 0.2 mL tube
    2. Reagent uL per Sample uL Total (Samples+NT) uL Total (No Phosphate) uL Total (No C Probe)
      PCCA-03 (10 uM) 1 4 2 0
      PCCB-03 (10 uM) 1 4 2*** 0
      Wash buffer 3 12 6 10
      Total 5 20 10 10
    3. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    4. Add 5 uL probe mixture to beads
    5. Incubate at 40C for 1 hour with agitation
    6. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant

Protocol-Phase 2[edit]

  1. Lock and Latch Hybridization/Ligation
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (8.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 32.8
      Latch Oligo 10 uM 10 pmol 1 8.2
      Lock Oligo 10 uM 10 pmol 1 8.2
      nfH2O NA NA 13 106.6
      Total     19 155.8
    3. Add 19 uL probe mix to each sample
    4. Incubate at 65C for 5 min; Note: changed from 95C due to this
    5. Lower temperature to 55C; incubate 20 minutes
    6. Lower the temperature to 20C; wait 30 seconds then take off incubator
    7. Add 1 uL T4 Ligase to each reaction
    8. Incubate at RT for 30 min
    9. Heat kill enzyme by incubating at 65C for 10 minutes
    10. Wash twice with 100 uL wash buffer
  2. Padlock Hybridization
    1. Make the following 8.1X master mix (1X)
      1. 16.2 uL 10 uM padlock0501(2uL)
      2. 16.2 uL Amp Ligase 10X reaction buffer(2uL)
      3. 129.6 uL nfH2O
      4. (16uL)
    2. Heat mixture to 95C for 5 minutes; Cooldown on ice
    3. Resuspend sample in 20 uL padlock buffer
    4. Incubate at 37C for 30 minutes
  3. Circularization
    1. Prepare 8.5X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.5x) (uL)
      NAD+ 5 mM 40 nmol 8 68
      dNTP 1 mM 600 pmol 0.6 5.1
      Betaine 5 M 15 umol 3 25.5
      10X AmpLigase Buffer 10X 1X 2 17
      Amp Ligase 5 U/uL 10 U 2 17
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 27.2
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL phusion mix to 20 uL reaction
    4. Incubate at 55C for 2 hours

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 master mix according to table below
    2. Reagent uL Added Master Mix (16.2X)
      Template 4 NA
      RCA Primer (10 uM) 2.5 40.5
      dNTP (1 mM) 2 32.4
      10X Buffer 2 32.4
      Phi29 1 NA
      BSA (10 mg/mL) 0.4 6.48
      nfH2O 8.1 131.22
      Total 20 243
    3. Add 14 uL master mix to each tube
    4. Add sample and to new reaction tubes according to following table
    5. Sample Code Phi29 Source Incubation Time (hrs) Sample (1AB) No Template (2AB) No C Probes (3AB) No PO4 (4AB)
      X NEB 3 hrs X X    
      Y NEB 6 hrs X   X  
      Z NEB 12 hrs X     X
      Q Epicentre 3 hrs X     X
    6. Incubate at 30C for X hours (see table above)
    7. Incubate at 65C for 10 minutes;Hold at 10C until next step


    Continued tomorrow