Daniel:Notebook/ComboLock/2016-12-16: Difference between revisions

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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:#92CDDC;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center"
| width="180" height="32" | Reagent
| width="160" height="30" | Reagent
| width="65" | uL Added
| width="85" | uL Added
| width="80" | Master Mix (7X)
| width="85" | Master Mix (13X)


|- style="font-size:12pt"
|- style="font-size:12pt"
Line 105: Line 105:
| height="15"  valign="bottom" | RCA Primer (100 uM)
| height="15"  valign="bottom" | RCA Primer (100 uM)
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 2.5
| align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 32.5


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | dNTP (1 mM)
| height="15"  valign="bottom" | dNTP (1 mM)
| align="center" align="center" valign="bottom" | 0.8
| align="center" align="center" valign="bottom" | 0.8
| align="center" align="center" valign="bottom" | 5.6
| align="center" align="center" valign="bottom" | 10.4


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X Buffer
| height="15"  valign="bottom" | 10X Buffer
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 14
| align="center" align="center" valign="bottom" | 26


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phi29
| height="15"  valign="bottom" | Phi29
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 7
| align="center" align="center" valign="bottom" | 13


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 8.7
| align="center" align="center" valign="bottom" | 8.7
| align="center" align="center" valign="bottom" | 60.9
| align="center" align="center" valign="bottom" | 113.1


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Total
| height="15"  valign="bottom" | Total
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20
| align="center" align="center" valign="bottom" | 105
|style="font-weight:bold" align="center" align="center" valign="bottom" | 195


|}
|}
Line 335: Line 335:
</ol>
</ol>


=Version 2 Oligos=
===Results===


The version 2 oligos have several changes. The first is that they are shorter. The C probes have a 20bp target matching region (from 24bp) and the adapters are 15bp (from 18bp). This makes the C probes exactly 60bp which means they are regular oligos (not Ultramers) and can be received one day after ordering. A second improvement is to include the idea of the [[Daniel:Notebook/ComboLock/2016-12-9|''lock oligo'']]. Note, this is not a [https://en.wikipedia.org/wiki/Locked_nucleic_acid Lock nucleic acid], it's just a name. The lock oligo fits in between the two C probes, spanning the gap between them. In conjunction with the latch oligo, this theoretically means that we can ligate the C probes together without using polymerase. This will allow the padlock probe to act as a standard gap-fill padlock (instead of 2 small gaps) since it is one medium sized gap. I won't have time today to actually run this, so the following protocol will only be for reference to start when I get back.
<gallery perrow=2 heights=300px widths=300px>
File:|qPCR curve
File:2016-12-16-MirrorCProbe-ONCap-Supernatant.png|Gel image-supernatant RCA
File:2016-12-16-MirrorCProbe-ONCap-Beads.png|Gel image-Beads RCA
</gallery>


==Sample Matrix==
==Adapter Testing==
 
Just like [[Daniel:Notebook/ComboLock/2016-12-15|yesterday]] I'm going to test presence of C probes and circular product in the pre-RCA mix.
 
<ol start="7">
<li>qPCR</li>
<ol type="A">
<li>Make master mix according to following recipes</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:#CCC0DA;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
| width="150" height="34" | Sample
| width="85" height="45" | Master Mix
| width="120" | Condition
| width="85" | Primers
| width="65" | Primer Target
| width="85" | 2X Kapa SYBR qPCR MM
| width="85" | 10 uM Forward Primer
| width="85" | 10 uM Reverse Primer
| width="65" | H2O
| width="65" | Master Mix


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 1 (AB)
|style="font-weight:bold" height="15"  | MMC1
| align="center" valign="bottom" | Normal
| align="center" | p24/p4*
| align="center" | C1
| align="center" align="center" | 230
| align="center" align="center" | 9.2
| align="center" align="center" | 9.2
| align="center" align="center" | 193.2
| align="center" | MMC1


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 2 (AB)
|style="font-weight:bold" height="15"  | MMC2
| align="center" valign="bottom" | No Template
| align="center" | p24/p4*
| align="center" | C1
| align="center" align="center" | 230
| align="center" align="center" | 9.2
| align="center" align="center" | 9.2
| align="center" align="center" | 193.2
| align="center" | MMC1


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 3 (AB)
|style="font-weight:bold" height="15"  | MMCP
| align="center" valign="bottom" | No C probes
| align="center" | p6/p12*
| align="center" | C2
| align="center" align="center" | 230
| align="center" align="center" | 9.2
| align="center" align="center" | 9.2
| align="center" align="center" | 193.2
| align="center" | MMC2
 
|}


|- style="background-color:#BFBFBF;font-size:12pt"
<li>Add 48 uL master mix to each well</li>
| height="15" valign="bottom" | Sample 4 (AB)
<li>Add 2 uL sample according to table in the following section</li>
| align="center" valign="bottom" | No phosphate activation
<li>qPCR Cycles</li>
<ol type="a">
<li>95C 3 min</li>
<li>95C 3 sec</li>
<li>55C 30 sec</li>
<li>72C 20 sec</li>
<li>plate read</li>
<li>goto b x24</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol></ol>
<li>TBE Gel</li>
<ol type="A">
<li>Mix 144 uL TBE, 36 uL 6x loading dye</li>
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li>
<li>Add 2 uL of sample or ladder to correct drop</li>
<li>Load 10 uL in to well</li>
<li>Run gel for 22 minutes at 250V</li>
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li>
<li>Rinse gel and image in gel doc</li>
</ol></ol>


|}
===Results===


==Protocol==
<gallery perrow=3 heights=300px widths=300px>
File:|qPCR curve displaying sample vs C probes (for supernatant)
File:|qPCR curve displaying sample vs C probes (for beads)
File:|qPCR curve displaying supernatant vs. beads effect
File:2016-12-16-MirrorCProbe-ONCap-Adaptors-CProbes.png|Gel image 1-Sample C probe detection
File:2016-12-16-MirrorCProbe-ONCap-Adaptors-Controls.png|Gel image 2-Controls gel
</gallery>


<ol>
===qPCR Sample Matrix===
<li>Phosphorylation-MirrorC Probe B (PCCB-02)</li>
<ol type="A">
<li>Set up reaction according to table</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:#C4D79B;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center"
| width="150" height="30" | Reagent
| width="100" height="45" | Sample
| width="92" | Stock Conc
| width="120" | Condition
| width="91" | Final Conc./Amount
| width="90" | Primers
| width="65" | uL added
| width="130" | Primer Target
| width="91" | Fraction
| width="65" | Lane
| width="65" | Sample Vol (uL)
| width="73" | 2X Kapa SYBR qPCR MM
| width="80" | 10 uM Forward Primer
| width="80" | 10 uM Reverse Primer
| width="65" | H2O
| width="65" | Total Volume (uL)
| width="65" | Master Mix


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="30"  valign="bottom" | T4 Polynucleotide Kinase Buffer
|style="font-weight:bold" height="30"  | Sample 1A
| align="center" | 10X
| align="center" | Sample
| align="center" | 1X
| align="center" | p24/p4*
| align="center" | C1
| align="center" | Supernatant-PreRCA
| align="center" | A1
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC1


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15valign="bottom" | ATP
|style="font-weight:bold" height="30| Sample 1B
| align="center" | 10 mM
| align="center" | Sample
| align="center" | 1 mM
| align="center" | p24/p4*
| align="center" | C1
| align="center" | Supernatant-PreRCA
| align="center" | A2
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC1


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15valign="bottom" | PCCB-02
|style="font-weight:bold" height="30| Sample 1A
| align="center" | 10 uM
| align="center" | Sample
| align="center" | 100 pmol total
| align="center" | p6/p12*
| align="center" align="center" | 10
| align="center" | C2
| align="center" | Supernatant-PreRCA
| align="center" | A3
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC2


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15valign="bottom" | T4 DNA Kinase
|style="font-weight:bold" height="30| Sample 1B
| align="center" | 10 U/uL
| align="center" | Sample
| align="center" | 10 U
| align="center" | p6/p12*
| align="center" | C2
| align="center" | Supernatant-PreRCA
| align="center" | A4
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC2


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15valign="bottom" | nfH2O
|style="font-weight:bold" height="30| Sample 1A
| align="center" | NA
| align="center" | Sample
| align="center" | NA
| align="center" | p24/p12*
| align="center" align="center" | 5
| align="center" | Circular Product
| align="center" | Supernatant-PreRCA
| align="center" | A5
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMCP


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15valign="bottom" | Total
|style="font-weight:bold" height="30| Sample 1B
| align="center" align="center" | &nbsp;
| align="center" | Sample
| align="center" align="center" | &nbsp;
| align="center" | p24/p12*
|style="font-weight:bold" align="center" align="center" | 20
| align="center" | Circular Product
| align="center" | Supernatant-PreRCA
| align="center" | A6
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMCP


|}
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 1A
| align="center" | Sample
| align="center" | p24/p4*
| align="center" | C1
| align="center" | Beads-PreRCA
| align="center" | C1
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC1


<li>Incubate at 37C for 30 min</li>
|- style="background-color:#DA9694;font-size:12pt"
<li>Heat kill enzyme with 15 minutes at 65C</li>
|style="font-weight:bold" height="15"  | Sample 1B
</ol>
| align="center" | Sample
<li>Template-Bead Binding</li>
| align="center" | p24/p4*
<ol type="A">
| align="center" | C1
<li>Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer </li>
| align="center" | Beads-PreRCA
<li>Apply magnet for 30 sec and remove supernatant</li>
| align="center" | C2
<li>Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min</li>
| align="center" align="center" | 2
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>
| align="center" align="center" | 25
</ol>
| align="center" align="center" | 1
<li>C Probe Hybridization</li>
| align="center" align="center" | 1
<ol type="A">
| align="center" align="center" | 21
<li>Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube</li>
| align="center" align="center" | 50
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
| align="center" | MMC1
<li>Add 5 uL probe mixture to beads</li>
<li>Incubate at 40C for 1 hour with agitation; '''start part 3 after incubation begins'''</li>
<li>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li>
</ol>
<li>Phosphorylation-Lock oligo</li>
<ol type="A">
<li>Set up reaction according to table</li>
 
{| 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="106" height="30" | Reagent
| width="92" | Stock Conc
| width="91" | Final Conc./Amount
| width="65" | uL added


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="30valign="bottom" | T4 DNA Ligase Buffer
|style="font-weight:bold" height="15| Sample 1A
| align="center" | 10X
| align="center" | Sample
| align="center" | 1X
| align="center" | p6/p12*
| align="center" | C2
| align="center" | Beads-PreRCA
| align="center" | C3
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC2


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#DA9694;font-size:12pt"
| height="15"  valign="bottom" | ATP
|style="font-weight:bold" height="15"  | Sample 1B
| align="center" | 10 mM
| align="center" | Sample
| align="center" | 1 mM
| align="center" | p6/p12*
| align="center" | C2
| align="center" | Beads-PreRCA
| align="center" | C4
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC2


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Lock0201
|style="font-weight:bold" height="15"  | Sample 1A
| align="center" | 10 uM
| align="center" | Sample
| align="center" | 10 pmol total
| align="center" | p24/p12*
| align="center" align="center" | 10
| align="center" | Circular Product
| align="center" | Beads-PreRCA
| align="center" | C5
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMCP


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#DA9694;font-size:12pt"
| height="15"  valign="bottom" | T4 DNA Kinase
|style="font-weight:bold" height="15"  | Sample 1B
| align="center" | 10 U/uL
| align="center" | Sample
| align="center" | 10 U
| align="center" | p24/p12*
| align="center" | Circular Product
| align="center" | Beads-PreRCA
| align="center" | C6
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMCP


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15valign="bottom" | nfH2O
|style="font-weight:bold" height="30| Sample 3A
| align="center" | NA
| align="center" | No C Probes
| align="center" | NA
| align="center" | p24/p4*
| align="center" align="center" | 5
| align="center" | C1
| align="center" | Supernatant-PreRCA
| align="center" | F1
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC1


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#C4D79B;font-size:12pt"
|style="font-weight:bold" height="15valign="bottom" | Total
|style="font-weight:bold" height="30| Sample 3B
| align="center" align="center" | &nbsp;
| align="center" | No C Probes
| align="center" align="center" | &nbsp;
| align="center" | p24/p4*
|style="font-weight:bold" align="center" align="center" | 20
| align="center" | C1
| align="center" | Supernatant-PreRCA
| align="center" | F2
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC1


|}
|- style="font-size:12pt"
|style="font-weight:bold" height="30"  | Sample 3A
| align="center" | No C Probes
| align="center" | p6/p12*
| align="center" | C2
| align="center" | Supernatant-PreRCA
| align="center" | F3
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC2


<li>Incubate at 37C for 30 min</li>
|- style="background-color:#C4D79B;font-size:12pt"
<li>Heat kill enzyme with 15 minutes at 65C</li>
|style="font-weight:bold" height="30"  | Sample 3B
</ol>
| align="center" | No C Probes
<li>Lock and Latch Hybridization/Ligation</li>
| align="center" | p6/p12*
<ol type="A">
| align="center" | C2
<li>Set up the following master mix</li>
| align="center" | Supernatant-PreRCA
 
| align="center" | F4
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
| align="center" align="center" | 2
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center"
| align="center" align="center" | 25
| width="114" height="30" | Reagent
| align="center" align="center" | 1
| width="65" | Stock Conc
| align="center" align="center" | 1
| width="69" | Final Conc./Amt
| align="center" align="center" | 21
| width="65" | uL added
| align="center" align="center" | 50
| width="65" | Master Mix (8.2X)
| align="center" | MMC2


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="30"  valign="bottom" | T4 Ligase Reaction Buffer
|style="font-weight:bold" height="30"  | Sample 3A
| align="center" | 10X
| align="center" | No C Probes
| align="center" | 1X
| align="center" | p24/p12*
| align="center" | Circular Product
| align="center" | Supernatant-PreRCA
| align="center" | F5
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" valign="bottom" | 16.4
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMCP


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#C4D79B;font-size:12pt"
| height="15valign="bottom" | Latch Oligo
|style="font-weight:bold" height="30| Sample 3B
| align="center" | 10 uM
| align="center" | No C Probes
| align="center" | 10 pmol
| align="center" | p24/p12*
| align="center" | Circular Product
| align="center" | Supernatant-PreRCA
| align="center" | F6
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" valign="bottom" | 8.2
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMCP


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="30valign="bottom" | Lock Oligo Phosphate Reaction
|style="font-weight:bold" height="15| Sample 3A
| align="center" | (5 uM Lock)
| align="center" | No C Probes
| align="center" | 10 pmol
| align="center" | p24/p4*
| align="center" | C1
| align="center" | Beads-PreRCA
| align="center" | H1
| align="center" align="center" | 2
| align="center" align="center" | 2
| align="center" align="center" valign="bottom" | 16.4
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC1


|- style="background-color:#BFBFBF;font-size:12pt"
|- style="background-color:#B1A0C7;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
|style="font-weight:bold" height="15"  | Sample 3B
| align="center" | NA
| align="center" | No C Probes
| align="center" | NA
| align="center" | p24/p4*
| align="center" align="center" | 14
| align="center" | C1
| align="center" align="center" valign="bottom" | 114.8
| align="center" | Beads-PreRCA
| align="center" | H2
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC1


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Total
|style="font-weight:bold" height="15"  | Sample 3A
| align="center" align="center" | &nbsp;
| align="center" | No C Probes
| align="center" align="center" | &nbsp;
| align="center" | p6/p12*
|style="font-weight:bold" align="center" align="center" | 19
| align="center" | C2
|style="font-weight:bold" align="center" align="center" valign="bottom" | 155.8
| align="center" | Beads-PreRCA
| align="center" | H3
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC2


|}
|- style="background-color:#B1A0C7;font-size:12pt"
|style="font-weight:bold" height="15"  | Sample 3B
| align="center" | No C Probes
| align="center" | p6/p12*
| align="center" | C2
| align="center" | Beads-PreRCA
| align="center" | H4
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 21
| align="center" align="center" | 50
| align="center" | MMC2


<li>Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201</li>
|- style="font-size:12pt"
<li>Incubate at 95C for 5 min</li>
|style="font-weight:bold" height="15"  | Sample 3A
<li>Lower the temperature to 20C; wait 30 seconds then take off incubator</li>
| align="center" | No C Probes
<li>Add 1 uL T4 Ligase to each reaction</li>
| align="center" | p24/p12*
<li>Incubate at RT for 30 min</li>
| align="center" | Circular Product
<li>Heat kill enzyme by incubating at 65C for 10 minutes</li>
| align="center" | Beads-PreRCA
<li>Wash twice with 100 uL wash buffer</li></ol>
| align="center" | H5
<li>Padlock Hybridization</li>
| align="center" align="center" | 2
<ol type="A">
| align="center" align="center" | 25
<li>Make following master mix</li>
| align="center" align="center" | 1
<ol type="a">
| align="center" align="center" | 1
<li>16.4 uL 10 uM padlock0401</li>
| align="center" align="center" | 21
<li>16.4 uL Amp Ligase 10X reaction buffer</li>
| align="center" align="center" | 50
<li>131.2 uL nfH2O</li></ol>
| align="center" | MMCP
<li>Resuspend sample in 20 uL padlock buffer</li>


<li>Circularization</li>
|- style="background-color:#B1A0C7;font-size:12pt"
<ol type="A">
|style="font-weight:bold" height="15"  | Sample 3B
<li>Prepare 9X uL Phusion mix</li>
| align="center" | No C Probes
 
| align="center" | p24/p12*
===Buffers===
| align="center" | Circular Product
 
| align="center" | Beads-PreRCA
'''Wash Buffer'''
| align="center" | H6
 
| align="center" align="center" | 2
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
| align="center" align="center" | 25
|- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center"
| align="center" align="center" | 1
| width="65" height="30" | Reagent
| align="center" align="center" | 1
| width="65" | Stock
| align="center" align="center" | 21
| width="65" | Final
| align="center" align="center" | 50
| width="65" | Dilution
| align="center" | MMCP
| width="65" | Amt in 20 mL


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | NaCl
|style="font-weight:bold" height="15"  valign="bottom" | NTC
| align="center" valign="bottom" | 1.5 M
| align="center" valign="bottom" | qPCR Primers Only
| align="center" valign="bottom" | 0.5 M
| align="center" valign="bottom" | p24/p4*
| align="center" align="center" valign="bottom" | 3
| align="center" valign="bottom" | C1
| align="center" valign="bottom" | 6.66 mL
| align="center" valign="bottom" | NA
| align="center" | A7
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 23
| align="center" align="center" | 50
| align="center" | MMC1


|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#FABF8F;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Tris-HCl
|style="font-weight:bold" height="15"  valign="bottom" | NTC
| align="center" valign="bottom" | 500 mM
| align="center" valign="bottom" | qPCR Primers Only
| align="center" valign="bottom" | 20 mM
| align="center" valign="bottom" | p6/p12*
| align="center" align="center" valign="bottom" | 25
| align="center" valign="bottom" | C2
| align="center" valign="bottom" | 800 uL
| align="center" valign="bottom" | NA
| align="center" | C7
| align="center" align="center" | 2
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 23
| align="center" align="center" | 50
| align="center" | MMC2


|- style="font-size:12pt"
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | EDTA
|style="font-weight:bold" height="15"  valign="bottom" | NTC
| align="center" valign="bottom" | 0.5M
| align="center" valign="bottom" | qPCR Primers Only
| align="center" valign="bottom" | 1 mM
| align="center" valign="bottom" | p24/p12*
| align="center" align="center" valign="bottom" | 500
| align="center" valign="bottom" | Circular Product
| align="center" valign="bottom" | 40 uL
 
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | nf H2O
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" | F7
| align="center" valign="bottom" | NA
| align="center" align="center" | 2
| align="center" valign="bottom" | 12.5 mL
| align="center" align="center" | 25
| align="center" align="center" | 1
| align="center" align="center" | 1
| align="center" align="center" | 23
| align="center" align="center" | 50
| align="center" | MMCP


|}
|}




[[Category:ComboLock]] [[Category:20161215]]
[[Category:ComboLock]] [[Category:20161215]]

Latest revision as of 01:15, 17 December 2016

Overnight Capture (Started Yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. Circularization
    1. Prepare 7X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (7x) (uL)
      NAD+ 5 mM 40 nmol 8 56
      dNTP 1 mM 600 pmol 0.6 4.2
      Betaine 5 M 15 umol 3 21
      10X AmpLigase Buffer 10X 1X 2 14
      Amp Ligase 5 U/uL 10 U 2 14
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 22.4
      nf H2O     1.2 8.4
      Total     20 140
    3. Add 20 uL Phusion mix to 20 uL of reaction
    4. Incubate at 60C for 2 hours
  2. Exonuclease Digestion
    1. Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
    2. Add 2 uL to each sample, mix by swirling pipette tip
    3. Incubate for 1 hour at 37C
    4. Heat kill by incubating for 5 min at 95C
  3. Rolling Circle Amplification
    1. Prepare 7X Master mix according to table below
    2. Add 15 uL master mix to each tube
    3. Add sample to new reaction tubes according to following table; use template from both supernatant only and beads
    4. Reagent uL Added Master Mix (13X)
      Template 5 NA
      RCA Primer (100 uM) 2.5 32.5
      dNTP (1 mM) 0.8 10.4
      10X Buffer 2 26
      Phi29 1 13
      nfH2O 8.7 113.1
      Total 20 195


    5. Incubate at 37C for 3 hours
    6. Incubate at 65C for 10 minutes;Hold at 10C until next step
  4. qPCR
    1. Make master mix according to following recipe
      1. 151.2 uL nfH2O
      2. 180 uL SYBR Fast
      3. 7.2 uL 10 uM AmpF
    2. Add 47 uL master mix to each well
    3. Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
    4. Sample Condition Fraction Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL)
      Sample 1A Sample Supernatant A1 20 2 25 1 1 21 50
      Sample 1B Sample Supernatant A2 20 2 25 1 1 21 50
      Sample 2A No Template Supernatant A3 21 2 25 1 1 21 50
      Sample 2B No Template Supernatant A4 21 2 25 1 1 21 50
      Sample 3A No C Probe Supernatant A5 22 2 25 1 1 21 50
      Sample 3B No C Probe Supernatant A6 22 2 25 1 1 21 50
      Sample 1A Sample Beads A1 20 2 25 1 1 21 50
      Sample 1B Sample Beads A2 20 2 25 1 1 21 50
      Sample 2A No Template Beads A3 21 2 25 1 1 21 50
      Sample 2B No Template Beads A4 21 2 25 1 1 21 50
      Sample 3A No C Probe Beads A5 22 2 25 1 1 21 50
      Sample 3B No C Probe Beads A6 22 2 25 1 1 21 50
      NTC qPCR Primers Only   A7 23 2 25 1 1 23 50

    Results[edit]

    Adapter Testing[edit]

    Just like yesterday I'm going to test presence of C probes and circular product in the pre-RCA mix.

    1. qPCR
      1. Make master mix according to following recipes
      2. Master Mix Primers Primer Target 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Master Mix
        MMC1 p24/p4* C1 230 9.2 9.2 193.2 MMC1
        MMC2 p24/p4* C1 230 9.2 9.2 193.2 MMC1
        MMCP p6/p12* C2 230 9.2 9.2 193.2 MMC2
      3. Add 48 uL master mix to each well
      4. Add 2 uL sample according to table in the following section
      5. qPCR Cycles
        1. 95C 3 min
        2. 95C 3 sec
        3. 55C 30 sec
        4. 72C 20 sec
        5. plate read
        6. goto b x24
        7. 72C 2 min
        8. 16C hold
    2. TBE Gel
      1. Mix 144 uL TBE, 36 uL 6x loading dye
      2. Aliquot 10 uL per sample/ladder lane onto parafilm
      3. Add 2 uL of sample or ladder to correct drop
      4. Load 10 uL in to well
      5. Run gel for 22 minutes at 250V
      6. Open gel and stain with 3 uL SYBR Gold for 3 minutes
      7. Rinse gel and image in gel doc

    Results[edit]

    qPCR Sample Matrix[edit]

    Sample Condition Primers Primer Target Fraction Lane Sample Vol (uL) 2X Kapa SYBR qPCR MM 10 uM Forward Primer 10 uM Reverse Primer H2O Total Volume (uL) Master Mix
    Sample 1A Sample p24/p4* C1 Supernatant-PreRCA A1 2 25 1 1 21 50 MMC1
    Sample 1B Sample p24/p4* C1 Supernatant-PreRCA A2 2 25 1 1 21 50 MMC1
    Sample 1A Sample p6/p12* C2 Supernatant-PreRCA A3 2 25 1 1 21 50 MMC2
    Sample 1B Sample p6/p12* C2 Supernatant-PreRCA A4 2 25 1 1 21 50 MMC2
    Sample 1A Sample p24/p12* Circular Product Supernatant-PreRCA A5 2 25 1 1 21 50 MMCP
    Sample 1B Sample p24/p12* Circular Product Supernatant-PreRCA A6 2 25 1 1 21 50 MMCP
    Sample 1A Sample p24/p4* C1 Beads-PreRCA C1 2 25 1 1 21 50 MMC1
    Sample 1B Sample p24/p4* C1 Beads-PreRCA C2 2 25 1 1 21 50 MMC1
    Sample 1A Sample p6/p12* C2 Beads-PreRCA C3 2 25 1 1 21 50 MMC2
    Sample 1B Sample p6/p12* C2 Beads-PreRCA C4 2 25 1 1 21 50 MMC2
    Sample 1A Sample p24/p12* Circular Product Beads-PreRCA C5 2 25 1 1 21 50 MMCP
    Sample 1B Sample p24/p12* Circular Product Beads-PreRCA C6 2 25 1 1 21 50 MMCP
    Sample 3A No C Probes p24/p4* C1 Supernatant-PreRCA F1 2 25 1 1 21 50 MMC1
    Sample 3B No C Probes p24/p4* C1 Supernatant-PreRCA F2 2 25 1 1 21 50 MMC1
    Sample 3A No C Probes p6/p12* C2 Supernatant-PreRCA F3 2 25 1 1 21 50 MMC2
    Sample 3B No C Probes p6/p12* C2 Supernatant-PreRCA F4 2 25 1 1 21 50 MMC2
    Sample 3A No C Probes p24/p12* Circular Product Supernatant-PreRCA F5 2 25 1 1 21 50 MMCP
    Sample 3B No C Probes p24/p12* Circular Product Supernatant-PreRCA F6 2 25 1 1 21 50 MMCP
    Sample 3A No C Probes p24/p4* C1 Beads-PreRCA H1 2 25 1 1 21 50 MMC1
    Sample 3B No C Probes p24/p4* C1 Beads-PreRCA H2 2 25 1 1 21 50 MMC1
    Sample 3A No C Probes p6/p12* C2 Beads-PreRCA H3 2 25 1 1 21 50 MMC2
    Sample 3B No C Probes p6/p12* C2 Beads-PreRCA H4 2 25 1 1 21 50 MMC2
    Sample 3A No C Probes p24/p12* Circular Product Beads-PreRCA H5 2 25 1 1 21 50 MMCP
    Sample 3B No C Probes p24/p12* Circular Product Beads-PreRCA H6 2 25 1 1 21 50 MMCP
    NTC qPCR Primers Only p24/p4* C1 NA A7 2 25 1 1 23 50 MMC1
    NTC qPCR Primers Only p6/p12* C2 NA C7 2 25 1 1 23 50 MMC2
    NTC qPCR Primers Only p24/p12* Circular Product NA F7 2 25 1 1 23 50 MMCP