Daniel:Notebook/ComboLock/2016-8-18: Difference between revisions

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>Djacobse
>Djacobse
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<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<ol type="A">
<li>Vacuum centrifuge samples to concentrate; resuspend in 2 uL</li>
<li>Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer (total)</li>
<li>Suspend 1 uL (4 ug) beads in 100 uL wash buffer</li>
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
| width="49" height="31" | Tube
| width="121" | Biotin-Amplicon
| width="54" | uL beads added
| width="105" | uL biotin probe added
 
|- style="font-size:12pt"
| align="center" height="15" valign="bottom" | 1
| align="center" valign="bottom" | Amp1
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 10
 
|- style="background-color:#D9D9D9;font-size:12pt"
| align="center" height="15" valign="bottom" | 2
| align="center" valign="bottom" | Amp3
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 10
 
|- style="font-size:12pt"
| align="center" height="30" valign="bottom" | 3
| align="center" valign="bottom" | PCAmp2 (C2)
| align="center" align="center" valign="bottom" | 1
| align="center" valign="bottom" | 2 (dilute to 1 pmol/uL)
 
|}
 
<li>Apply magnet for 30 sec and remove supernatant</li>
<li>Apply magnet for 30 sec and remove supernatant</li>
<li>Add 2 uL oligo to bead solution; incubate at RT for 5 min</li>
<li>Add 2 uL oligo per sample to bead solution (see above table); incubate at RT for 5 min</li>
<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>Mix together samples according to table in 0.2 mL tubes; use 100 uL LPH buffer with 100 nM Padlock0001-68 </li>
<li>Mix together samples according to table in 0.2 mL tubes; use Padlock0001-68 </li>
<li>Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes</li>
<li>Chill on ice for 3 minutes</li>
<li>Add sample to prepared strep beads; vortex to suspend</li>
<li>Incubate for 30 min at 37C</li>
<li>Apply magnet and remove supernatant</li>
<li>Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant</li>
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol>
<li>Circularization</li>
<ol type="A">
<li>Prepare 20 uL KLN mix</li>
<li>Prepare 4.1x master mix of Phusion mix</li>
<li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</li>
<li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li>
<li>Incubate at 55C for 4 hours </li>
<li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li>
</ol>
<li>Elution</li>
<ol type="A">
<li>Add 25 uL prewarmed elution buffer; vortex to suspend</li>
<li>Incubate at RT for 2 minutes</li>
<li>Apply magnet and transfer supernatant to a clean centrifuge tube</li>
<li>Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution</li>
</ol>


{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
| width="42" height="31" | Sample No
| width="49" height="31" | Sample No
| width="121" | Sample Name
| width="121" | Sample Name
| width="54" | LatchX2
| width="95" | LatchX2 (300 ng - 2 uL 10 uM)
| width="53" | LatchX3
| width="94" | LatchX3 (300 ng - 2 uL 10 uM)
| width="54" | PCAmp1
| width="54" | PCAmp1
| width="51" | PCAmp3
| width="51" | PCAmp3
Line 266: Line 268:
|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#D9D9D9;font-size:12pt"
| align="center" height="15" valign="bottom" | 8
| align="center" height="15" valign="bottom" | 8
|  valign="bottom" | C2 Only
|  valign="bottom" | PCAmp2 Only
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
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|}
|}
<li>Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes</li>
<li>Chill on ice for 3 minutes</li>
<li>Add sample to prepared strep beads; vortex to suspend</li>
<li>Incubate for 30 min at 37C</li>
<li>Apply magnet and remove supernatant</li>
<li>Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant</li>
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol>
<li>Circularization</li>
<ol type="A">
<li>Prepare 20 uL KLN mix</li>
<li>Prepare 4.1x master mix of Phusion mix</li>
<li>Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5</li>
<li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li>
<li>Incubate at 55C for 4 hours </li>
<li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li>
</ol>
<li>Elution</li>
<ol type="A">
<li>Add 25 uL prewarmed elution buffer; vortex to suspend</li>
<li>Incubate at RT for 2 minutes</li>
<li>Apply magnet and transfer supernatant to a clean centrifuge tube</li>
<li>Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution</li>
</ol>
[[Category:ComboLock]] [[Category:20160818]]
[[Category:ComboLock]] [[Category:20160818]]

Revision as of 18:26, 18 August 2016

Positive Control Amplicon Test

Back to Calendar

Buffer Prep

Wash/Binding Buffer

Reagent Stock Final Dilution Amt in 10 mL
NaCl 1.5 M 0.5 M 3 3.33 mL
Tris-HCl 500 mM 20 mM 25 400 uL
EDTA 0.5M 1 mM 500 20 uL
nf H2O NA NA NA 6.25 mL

Elution Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
Tris-HCl 500 mM 10 mM 50 20
EDTA 0.5M 1 mM 500 2
nf H2O NA NA NA 978

Low Salt Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
NaCl 1.5 M 0.15 M 10 100
Tris-HCl 500 mM 20 mM 25 40
EDTA 0.5M 1 mM 500 2
nf H2O NA NA NA 858

KLN Mix

Reagent Stock uL added
Amp Ligase Buffer 10X 2
dNTPs 100 uM 2
Hemo Klentaq NA 4
Amp Ligase 5 U/uL 2
nfH2O NA 10

Protocol

  1. Latch and Padlock Hybridization
    1. Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer (total)
    2. Tube Biotin-Amplicon uL beads added uL biotin probe added
      1 Amp1 5 10
      2 Amp3 5 10
      3 PCAmp2 (C2) 1 2 (dilute to 1 pmol/uL)
    3. Apply magnet for 30 sec and remove supernatant
    4. Add 2 uL oligo per sample to bead solution (see above table); incubate at RT for 5 min
    5. Apply magnet for 30 sec and remove supernatant
    6. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    7. Mix together samples according to table in 0.2 mL tubes; use Padlock0001-68
    8. Sample No Sample Name LatchX2 (300 ng - 2 uL 10 uM) LatchX3 (300 ng - 2 uL 10 uM) PCAmp1 PCAmp3 Expected Product
      1 Amp1 Test X   X   Amp1
      2 Amp3 Test   X   X Amp3
      3 LatchX2 Specificity X   X X Amp1
      4 LatchX3 Specificity   X X X Amp3
      5 No Template X X     None
      6 No Latch     X X None
      7 No Padlock X X X X None
      8 PCAmp2 Only X X     None
    9. Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
    10. Chill on ice for 3 minutes
    11. Add sample to prepared strep beads; vortex to suspend
    12. Incubate for 30 min at 37C
    13. Apply magnet and remove supernatant
    14. Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
    15. Repeat wash step above
    16. Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
    17. Resuspend with 20 uL 1x Amp Ligase buffer
  2. Circularization
    1. Prepare 20 uL KLN mix
    2. Prepare 4.1x master mix of Phusion mix
    3. Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
    4. Add 2 uL KLN mix to 20 uL of reaction for sample 1
    5. Incubate at 55C for 4 hours
    6. Heat inactivate enzyme by incubating for 2 minutes at 94C
  3. Elution
    1. Add 25 uL prewarmed elution buffer; vortex to suspend
    2. Incubate at RT for 2 minutes
    3. Apply magnet and transfer supernatant to a clean centrifuge tube
    4. Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution