Daniel:Notebook/PosSequencing/2017-6-21: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=Bead qPCR Test= Back to Calendar ==Protocol==") |
>Djacobse |
||
Line 4: | Line 4: | ||
==Protocol== | ==Protocol== | ||
<ol> | |||
<li>Template-Bead Binding; Make 4 samples and follow this protocol for each</li> | |||
<ol type="A"> | |||
<li>Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer</li> | |||
<li>Apply magnet for 30 sec and remove supernatant</li> | |||
<li>Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)</li> | |||
<li>Add 5 uL 10 uM (50 pmol total, 2X excess) dual biotin oligo and 15 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li> | |||
<li>Wash twice with 100 uL Dynabuffer</li> | |||
<li>Resuspend beads in 10 uL TE buffer</li> | |||
</ol> | |||
<li>Dilutions</li> | |||
<ol type="A"> | |||
<li>Make dilutions using 2 uL suspended beads with 18 uL TE for dilutions</li> | |||
<li>Make dilutions of 1:10, 1:100, 1:1000, and 1:10000</li> | |||
'''Sample Matrix''' | |||
{| 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="100" height="40" | Beads in Sample | |||
| width="95" | Dilution (Power 10) | |||
| width="95" | Amount Added (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 10 M | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 2 M | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 1 M | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 0.2 M | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 0.1 M | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 20 K | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 10 K | |||
| align="center" align="center" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 2 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 1 K | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 2 | |||
|} | |||
</ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make the following master mix</li> |
Revision as of 15:56, 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
Beads in Sample | Dilution (Power 10) | Amount Added (uL) |
10 M | 0 | 2 |
2 M | 1 | 4 |
1 M | 1 | 2 |
0.2 M | 2 | 4 |
0.1 M | 2 | 2 |
20 K | 3 | 4 |
10 K | 3 | 2 |
1 K | 4 | 2 |