Daniel:Notebook/ComboLock/2016-8-18: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
(4 intermediate revisions by the same user not shown) | |||
Line 150: | Line 150: | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
| align="center" align="center" valign="bottom" | 10 | | align="center" align="center" valign="bottom" | 10 | ||
|} | |||
==Buffers== | |||
'''KLN Mix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |||
| width="187" height="40" | Reagent | |||
| width="65" | Stock | |||
| width="65" | uL added | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase Buffer | |||
| align="center" valign="bottom" | 10X | |||
| align="center" align="center" valign="bottom" | 3 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | dNTPs | |||
| align="center" valign="bottom" | 100 uM | |||
| align="center" align="center" valign="bottom" | 3 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Hemo Klentaq | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 6 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Amp Ligase | |||
| align="center" valign="bottom" | 5 U/uL | |||
| align="center" align="center" valign="bottom" | 3 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 15 | |||
|} | |} | ||
Line 158: | Line 195: | ||
<li>Latch and Padlock Hybridization</li> | <li>Latch and Padlock Hybridization</li> | ||
<ol type="A"> | <ol type="A"> | ||
<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 | <li>Mix together samples according to table in 0.2 mL tubes; use Padlock0001-68 </li> | ||
{| 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=" | | width="49" height="31" | Sample No | ||
| width=" | | width="180" | Sample Name | ||
| width=" | | width="95" | LatchX2 (300 ng - 2 uL 10 uM) | ||
| width=" | | width="94" | LatchX3 (300 ng - 2 uL 10 uM) | ||
| width="54" | PCAmp1 | | width="54" | PCAmp1 | ||
| width="51" | PCAmp3 | | width="51" | PCAmp3 | ||
Line 262: | Line 301: | ||
| align="center" valign="bottom" | X | | align="center" valign="bottom" | X | ||
| align="center" valign="bottom" | X | | align="center" valign="bottom" | X | ||
| align="center" valign="bottom" | None | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 8 | |||
| valign="bottom" | PCAmp2 Only | |||
| align="center" valign="bottom" | X | |||
| align="center" valign="bottom" | X | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" valign="bottom" | | |||
| align="center" valign="bottom" | None | | align="center" valign="bottom" | None | ||
|} | |} | ||
<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 overnight; continued [[Daniel:Notebook/ComboLock/2016-8-19|tomorrow]] </li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20160818]] | [[Category:ComboLock]] [[Category:20160818]] |
Latest revision as of 22:30, 22 August 2016
Positive Control Amplicon Test[edit]
Buffer Prep[edit]
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 |
Buffers[edit]
KLN Mix
Reagent | Stock | uL added |
Amp Ligase Buffer | 10X | 3 |
dNTPs | 100 uM | 3 |
Hemo Klentaq | NA | 6 |
Amp Ligase | 5 U/uL | 3 |
nfH2O | NA | 15 |
Protocol[edit]
- Latch and Padlock Hybridization
- Suspend 1 uL (4 ug) beads per sample in 100 uL wash buffer (total)
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL oligo per sample to bead solution (see above table); incubate at RT for 5 min
- Apply magnet for 30 sec and remove supernatant
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Mix together samples according to table in 0.2 mL tubes; use Padlock0001-68
- Dissolve target in 50 uL wash buffer and heat at 65C for 5 minutes
- Chill on ice for 3 minutes
- Add sample to prepared strep beads; vortex to suspend
- Incubate for 30 min at 37C
- Apply magnet and remove supernatant
- Add 100 uL wash buffer and vortex to suspend beads; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer and vortex to suspend; apply magnet and remove supernatant
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Prepare 4.1x master mix of Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction for samples 2-5
- Add 2 uL KLN mix to 20 uL of reaction for sample 1
- Incubate at 55C overnight; continued tomorrow
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) |
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 |