Daniel:Notebook/PosSequencing/2017-6-21: Difference between revisions
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>Djacobse |
>Djacobse |
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{| 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:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="59" height="40" | Beads in Sample | ||
| width=" | | width="65" | Dilution (Power 10) | ||
| width=" | | width="65" | Amount Added (uL) | ||
| width="85" | ~Amt BioU Primer (pmol) | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 10 M | | height="15" valign="bottom" | 10 M | ||
| align=" | | align="right" align="center" valign="bottom" | 0 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" valign="bottom" | 5 pmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 2 M | | height="15" valign="bottom" | 2 M | ||
| align=" | | align="right" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 4 | ||
| align="center" valign="bottom" | 1 pmol | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 1 M | | height="15" valign="bottom" | 1 M | ||
| align=" | | align="right" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" valign="bottom" | 500 nmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 0.2 M | | height="15" valign="bottom" | 0.2 M | ||
| align=" | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 4 | ||
| align="center" valign="bottom" | 100 nmol | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 0.1 M | | height="15" valign="bottom" | 0.1 M | ||
| align=" | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" valign="bottom" | 50 nmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 20 K | | height="15" valign="bottom" | 20 K | ||
| align=" | | align="right" align="center" valign="bottom" | 3 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 4 | ||
| align="center" valign="bottom" | 10 nmol | |||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 10 K | | height="15" valign="bottom" | 10 K | ||
| align=" | | align="right" align="center" valign="bottom" | 3 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" valign="bottom" | 5 nmol | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 1 K | | height="15" valign="bottom" | 1 K | ||
| align=" | | align="right" align="center" valign="bottom" | 4 | ||
| align="center" align="center" valign="bottom" | | | align="right" align="center" valign="bottom" | 2 | ||
| align="center" valign="bottom" | 1 nmol | |||
|} | |} | ||
Line 76: | Line 84: | ||
<ol type="A"> | <ol type="A"> | ||
<li>Make the following master mix</li> | <li>Make the following master mix</li> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#95B3D7;font-size:12pt;font-weight:bold" align="center" | |||
| width="132" height="40" | Reagent | |||
| width="65" | 1X Added | |||
| width="65" | Master Mix (20.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Primer 2 (10 uM) | |||
| align="right" align="center" valign="bottom" | 4 | |||
| align="right" align="center" valign="bottom" | 80.8 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Barcode Oligo 100 pM | |||
| align="right" align="center" valign="bottom" | 5 | |||
| align="right" align="center" valign="bottom" | 101 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Beads | |||
| align="right" align="center" valign="bottom" | 4 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Kapa 2X SYBR Fast Mix | |||
| align="right" align="center" valign="bottom" | 20 | |||
| align="right" align="center" valign="bottom" | 404 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="right" align="center" valign="bottom" | 7 | |||
| align="right" align="center" valign="bottom" | 141.4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
| align="right" align="center" valign="bottom" | 40 | |||
| align="right" align="center" valign="bottom" | 727.2 | |||
|} | |||
<li>Add 36 uL master mix to each lane</li> | |||
<li>Add n uL of sample and 4-n uL nfH<sub>2</sub>O to appropriate lanes according to plate layout</li> | |||
[[Image:PlateLayout-BeadsqPCR-20170621.png|350x250px]] | |||
<li>Vortex and spin down after sealing wells</li> | |||
<li>Use the [[Daniel:Notebook/Protocols/1PCR|standard]] thermocycler program->40 cycles</li> | |||
</ol></ol> |
Revision as of 16:14, 21 June 2017
Bead qPCR Test
Protocol
- Template-Bead Binding; Make 4 samples and follow this protocol for each
- Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
- Add 5 uL 10 uM (50 pmol total, 2X excess) dual biotin oligo and 15 uL nfH2O per sample to bead solution; incubate at RT for 15 min
- Wash twice with 100 uL Dynabuffer
- Resuspend beads in 10 uL TE buffer
- Dilutions
- Make dilutions using 2 uL suspended beads with 18 uL TE for dilutions
- Make dilutions of 1:10, 1:100, 1:1000, and 1:10000 Sample Matrix
- qPCR
- Make the following master mix
- Add 36 uL master mix to each lane
- Add n uL of sample and 4-n uL nfH2O to appropriate lanes according to plate layout File:PlateLayout-BeadsqPCR-20170621.png
- Vortex and spin down after sealing wells
- Use the standard thermocycler program->40 cycles
Beads in Sample | Dilution (Power 10) | Amount Added (uL) | ~Amt BioU Primer (pmol) |
10 M | 0 | 2 | 5 pmol |
2 M | 1 | 4 | 1 pmol |
1 M | 1 | 2 | 500 nmol |
0.2 M | 2 | 4 | 100 nmol |
0.1 M | 2 | 2 | 50 nmol |
20 K | 3 | 4 | 10 nmol |
10 K | 3 | 2 | 5 nmol |
1 K | 4 | 2 | 1 nmol |
Reagent | 1X Added | Master Mix (20.2X) |
Primer 2 (10 uM) | 4 | 80.8 |
Barcode Oligo 100 pM | 5 | 101 |
Beads | 4 | 0 |
Kapa 2X SYBR Fast Mix | 20 | 404 |
nfH2O | 7 | 141.4 |
Total | 40 | 727.2 |