Daniel:Notebook/PosSequencing/2017-6-21: Difference between revisions

From ZhangLabWiki
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

Back to Calendar

Protocol

  1. Template-Bead Binding; Make 4 samples and follow this protocol for each
    1. Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
    4. 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
    5. Wash twice with 100 uL Dynabuffer
    6. Resuspend beads in 10 uL TE buffer
  2. Dilutions
    1. Make dilutions using 2 uL suspended beads with 18 uL TE for dilutions
    2. Make dilutions of 1:10, 1:100, 1:1000, and 1:10000
    3. Sample Matrix
      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


  3. qPCR
    1. Make the following master mix