Daniel:Notebook/ComboLock/2017-1-23: Difference between revisions

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==Protocol==


==Protocol==
==Protocol==


<ol>
<ol>
<li>Phosphorylation</li>
<ol type="A">
<li>In a 0.2 mL tube, add ingredients according to table</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center"
| width="153" height="36" | Reagent
| width="73" | Stock Conc
| width="90" | Final Conc./Amount
| width="65" | uL added
|- style="font-size:12pt"
| height="15"  valign="bottom" | AmpLigase Reaction Buffer
| align="center" | 10X
| align="center" | 1X
| align="center" align="center" | 2
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | ATP
| align="center" | 10 mM
| align="center" | 1 mM
| align="center" align="center" | 2
|- style="font-size:12pt"
| height="15"  valign="bottom" | PCAmp2
| align="center" | 100 uM
| align="center" | 1 nmol total
| align="center" align="center" | 10
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | T4 DNA Kinase
| align="center" | 10 U/uL
| align="center" | 10 U
| align="center" align="center" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" | NA
| align="center" | NA
| align="center" align="center" | 5
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Total
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
|style="font-weight:bold" align="center" align="center" | 20
|}
<li>Incubate at 37C for 30 min</li>
</ol>
<li>Ligation</li>
<ol type="A">
<li>Set up 2 reactions with the following reagents (DO NOT ADD LIGASE YET)</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center"
| width="162" height="45" | Reagent
| width="71" | Stock Conc
| width="79" | Final Conc./Amount
| width="65" | uL added
|- style="font-size:12pt"
| height="15"  valign="bottom" | AmpLigase Reaction Buffer
| align="center" | 10X
| align="center" | 1X
| align="center" align="center" | 4
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Latch Oligo
| align="center" | 100 uM
| align="center" | 500 umol
| align="center" align="center" | 5
|- style="font-size:12pt"
| height="15"  valign="bottom" | 5' Amplicon Oligo
| align="center" | 100 uM
| align="center" | 500 umol
| align="center" align="center" | 5
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Amp Ligase
| align="center" | 5 U/uL
| align="center" | 5 U
| align="center" align="center" | 1
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phosphate Reaction
| align="center" | NA
| align="center" | NA
| align="center" align="center" | 10
|- style="font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" | NA
| align="center" | NA
| align="center" align="center" | 25
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Total
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
|style="font-weight:bold" align="center" align="center" | 50
|}
<li>Heat reaction to 95C for 5 min</li>
<li>Lower temp to 55C</li>
<li>Add 1 uL Amp Ligase to each reaction without removing from thermocycler; swirl with pipette tip 5 times to mix</li>
<li>Incubate at 55C for 2 hours</li>
<li>Heat to 95 C to denature dsDNA
<li>Purify with ssDNA column</li>
</ol>
<li>ssDNA Column</li>
<ol type="A">
<li>Add 100 uL '''Binding Buffer''' to the sample; mix well</li>
<li>Transfer to '''IIC Column''' and centrifuge at 14000 rpm for 1 minute; '''SAVE THE FLOW THROUGH'''</li>
<li>Add 150 uL 100% EtOH to flow through; mix well</li>
<li>Transfer to '''IC Column''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li>
<li>Add 400 uL '''Prep Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li>
<li>Add 700 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li>
<li>Add 400 uL '''Wash Buffer''' and centrifuge at 14000 rpm for 1 minute; discard flow through</li>
<li>Centrifuge empty column at 14000 rpm for 2 minutes</li>
<li>Transfer to empty 1.5mL centrifuge tube (low bind)</li>
<li>Add 30 uL nfH2O and centrifuge at 14000 rpm for 1 minute</li>
</ol>
******
<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<ol type="A">
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<li>Apply magnet for 30 sec and remove supernatant</li>
<li>Apply magnet for 30 sec and remove supernatant</li>
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>
<li>Add 2 uL per sample of 10 uM Padlock0501 to 20 uL 1X Amp ligase buffer</li>
<li>Prep 8.2X Padlock0501 mixture buffer</li>
<ol type="a">
<li>16.4 uL 10 uM Padlock0501</li>
<li>16.4 uL 10X Amp Ligase Buffer</li>
<li>131.2 uL nfH2O</li></ol>
<li>Heat probes to 95C for 5 minutes</li>
<li>Heat probes to 95C for 5 minutes</li>
<li>Chill on ice for 3 minutes</li>
<li>Chill on ice for 3 minutes</li>
Line 252: Line 128:
<li>Incubate at 55C for 4 hours</li>
<li>Incubate at 55C for 4 hours</li>
</ol>
</ol>
Continued [[Daniel:Notebook/ComboLock/2017-1-24|tomorrow]]
[[Category:ComboLock]] [[Category:20170123]]

Latest revision as of 23:57, 24 January 2017

Circularization Test[edit]

Back to Calendar

Since the results from the last experiment seemed to indicate that RCA hasn't been working for me, I'm going to try RCA again using the old positive amplicon systems. For examples see Oct-5-2016 and Sept-30-2016. I'll be using PCAmp4

Sample Matrix[edit]

Tube No Molecules Available Amount
0 (AB) 1.00E+11 1 pmol
2 (AB) 1.00E+09 10 fmol
4 (AB) 1.00E+07 100 amol
6 (AB) 1.00E+05 1 amol

Protocol[edit]

  1. Latch and Padlock Hybridization
    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 oligo per sample to bead solution; incubate at RT for 5 min
    4. Apply magnet for 30 sec and remove supernatant
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Prep 8.2X Padlock0501 mixture buffer
      1. 16.4 uL 10 uM Padlock0501
      2. 16.4 uL 10X Amp Ligase Buffer
      3. 131.2 uL nfH2O
    7. Heat probes to 95C for 5 minutes
    8. Chill on ice for 3 minutes
    9. Add sample to prepared strep beads; vortex to suspend
    10. Incubate for 30 min at 37C with vigorous shaking
  2. 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 4 hours

    Continued tomorrow