Daniel:Notebook/ComboLock/2016-11-29: Difference between revisions

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(Created page with "=Extended Latch Test= Back to Calendar The purpose of this experiment is to try and get the circular product. We've had some difficulty with th...")
 
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The purpose of this experiment is to try and get the circular product. We've had some difficulty with the product containing more of the C-probe (see [[Daniel:Notebook/ComboLock/2016-11-18|Nov 18]] and [[Daniel:Notebook/ComboLock/2016-11-21|Nov 21]] results) sequence than they should. So I'm going to try an extended latch that covers the C1 barcode region besides the adapters and UMI. I can give the padlock a 5' phosphate group and then add only Amp ligase. This attach the latch to the padlock at locations where both are present. I can then gap fill.
The purpose of this experiment is to try and get the circular product. We've had some difficulty with the product containing more of the C-probe (see [[Daniel:Notebook/ComboLock/2016-11-18|Nov 18]] and [[Daniel:Notebook/ComboLock/2016-11-21|Nov 21]] results) sequence than they should. So I'm going to try an extended latch that covers the C1 barcode region besides the adapters and UMI. I can give the padlock a 5' phosphate group and then add only Amp ligase. This attach the latch to the padlock at locations where both are present. I can then gap fill.
===Sample Matrix===
{| 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"
| width="150" height="34" | Sample
| width="120" | Condition
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 1 (AB)
| align="center" valign="bottom" | Normal
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 2 (AB)
| align="center" valign="bottom" | No Template
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 3 (AB)
| align="center" valign="bottom" | No phosphate activation
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Sample 4 (AB)
| align="center" valign="bottom" | No C Probe
|}


==Protocol==
==Protocol==
Line 20: Line 45:
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add 5 uL probe mixture to beads</li>
<li>Add 5 uL probe mixture to beads</li>
<li>Incubate at 40C for 1 hour with agitation</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>Wash beads with 100 uL wash buffer; apply magnet and remove supernatant</li>
<li>Repeat wash step above</li>
<li>Repeat wash step above</li>
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li>
<li>Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant</li>
</ol>
</ol>
<ol>
<li>Phosphorylation</li>
<li>Phosphorylation</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>
Line 49: Line 75:
|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Padlock0201
| height="15"  valign="bottom" | Padlock0201
| align="center" | 15 uM
| align="center" | 10 uM
| align="center" | 150 pmol total
| align="center" | 150 pmol total
| align="center" align="center" | 15
| align="center" align="center" | 15
Line 73: Line 99:
|}
|}


<ol type="A">
<li>Incubate at 37C for 30 min</li>
<li>In a 0.2 mL tube, add ingredients according to table</li>
<li>Heat kill enzyme with 15 minutes at 65C</li>
</ol>
<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<ol type="A">
<li>Add following to a fresh 0.2 mL tube</li>
<li>Set up the following master mix</li>
<ol type="a">
 
<li>12.2 uL Latch0001, 10 uM stock</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
<li>12.2 uL Padlock0201, 10 uM stock</li>
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center"
<li>12.2 uL Amp10X buffer</li>
| width="180" height="30" | Reagent
<li>85.4 uL nfH2O</li></ol>
| width="85" | Stock Conc
<li>Add 20 uL probe mix to each sample; incubate 20C for 4 min</li>
| width="100" | Final Conc./Amount
| width="85" | uL added
| width="85" | Master Mix (6.2X)
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | AmpLigase Reaction Buffer
| align="center" | 10X
| align="center" | 1X
| align="center" align="center" | 2
| align="center" align="center" valign="bottom" | 12.4
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Latch Oligo
| align="center" | 10 uM
| align="center" | 20 pmol
| align="center" align="center" | 2
| align="center" align="center" valign="bottom" | 12.4
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Phosphate Reaction
| align="center" | NA
| align="center" | NA
| align="center" align="center" | 2
| align="center" align="center" valign="bottom" | 12.4
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" | NA
| align="center" | NA
| align="center" align="center" | 13
| align="center" align="center" valign="bottom" | 80.6
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Total
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
|style="font-weight:bold" align="center" align="center" | 19
|style="font-weight:bold" align="center" align="center" valign="bottom" | 117.8
 
|}
 
<li>Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201</li>
<li>Incubate at 95C for 5 min</li>
<li>Incubate at 95C for 5 min</li>
<li>Incubate at 60C for 1.5 hours</li></ol>
<li>Lower the temperature to 55C</li>
<li>Without removing the reaction from the incubator add 1 uL Amp Ligase to each reaction</li>
<li>Incubate at 55C for 2 hours</li></ol>
<li>Circularization</li>
<li>Circularization</li>
<ol type="A">
<ol type="A">
<li>Prepare 7X uL Phusion mix</li>
<li>Prepare 9X uL Phusion mix</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="150" height="30" | Reagent
| width="90" | Stock Conc
| width="90" | Final Amount
| width="90" | 1x Vol (uL)
| width="90" | MM Vol (9x) (uL)
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | NAD+
| align="center" | 5 mM
| align="center" | 40 nmol
| align="center" align="center" | 8
| align="center" align="center" | 72
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | dNTP
| align="center" | 1 mM
| align="center" | 600 pmol
| align="center" align="center" | 0.6
| align="center" align="center" | 5.4
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Betaine
| align="center" | 5 M
| align="center" | 15 umol
| align="center" align="center" | 3
| align="center" align="center" | 27
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 10X AmpLigase Buffer
| align="center" | 10X
| align="center" | 1X
| align="center" align="center" | 2
| align="center" align="center" | 18
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Amp Ligase
| align="center" | 5 U/uL
| align="center" | 10 U
| align="center" align="center" | 1
| align="center" align="center" | 9
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="30"  valign="bottom" | Phusion HF DNA Polymerase
| align="center" | 2000 U/mL
| align="center" | 6.4U
| align="center" align="center" | 4.2
| align="center" align="center" | 37.8
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | nf H2O
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
| align="center" align="center" | 1.2
| align="center" align="center" | 10.8
 
|- style="background-color:#BFBFBF;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Total
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
|style="font-weight:bold" align="center" align="center" | 20
|style="font-weight:bold" align="center" align="center" | 180
 
|}
 
<li>Add 20 uL Phusion mix to 20 uL of reaction</li>
<li>Add 20 uL Phusion mix to 20 uL of reaction</li>
<li>Incubate at 60C for 2 hours</li></ol>
<li>Incubate at 60C for 2 hours</li></ol>
</ol>
</ol>
Continued [[Daniel:Notebook/ComboLock/2016-11-30|tomorrow]]
[[Category:ComboLock]] [[Category:20161129]]

Latest revision as of 23:15, 1 December 2016

Extended Latch Test[edit]

Back to Calendar

The purpose of this experiment is to try and get the circular product. We've had some difficulty with the product containing more of the C-probe (see Nov 18 and Nov 21 results) sequence than they should. So I'm going to try an extended latch that covers the C1 barcode region besides the adapters and UMI. I can give the padlock a 5' phosphate group and then add only Amp ligase. This attach the latch to the padlock at locations where both are present. I can then gap fill.

Sample Matrix[edit]

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No phosphate activation
Sample 4 (AB) No C Probe

Protocol[edit]

  1. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
    2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    3. Add 5 uL probe mixture to beads
    4. Incubate at 40C for 1 hour with agitation; start part 3 after incubation begins
    5. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
  3. Phosphorylation
    1. Set up reaction according to table
    2. Reagent Stock Conc Final Conc./Amount uL added
      AmpLigase Reaction Buffer 10X 1X 2
      ATP 10 mM 1 mM 2
      Padlock0201 10 uM 150 pmol total 15
      T4 DNA Kinase 10 U/uL 10 U 1
      nfH2O NA NA 0
      Total     20
    3. Incubate at 37C for 30 min
    4. Heat kill enzyme with 15 minutes at 65C
  4. Latch and Padlock Hybridization
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amount uL added Master Mix (6.2X)
      AmpLigase Reaction Buffer 10X 1X 2 12.4
      Latch Oligo 10 uM 20 pmol 2 12.4
      Phosphate Reaction NA NA 2 12.4
      nfH2O NA NA 13 80.6
      Total     19 117.8
    3. Add 19 uL probe mix to each sample; for sample 3 don't add activated Padlock, add normal padlock0201
    4. Incubate at 95C for 5 min
    5. Lower the temperature to 55C
    6. Without removing the reaction from the incubator add 1 uL Amp Ligase to each reaction
    7. Incubate at 55C for 2 hours
  5. Circularization
    1. Prepare 9X uL Phusion mix
    2. Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (9x) (uL)
      NAD+ 5 mM 40 nmol 8 72
      dNTP 1 mM 600 pmol 0.6 5.4
      Betaine 5 M 15 umol 3 27
      10X AmpLigase Buffer 10X 1X 2 18
      Amp Ligase 5 U/uL 10 U 1 9
      Phusion HF DNA Polymerase 2000 U/mL 6.4U 4.2 37.8
      nf H2O     1.2 10.8
      Total     20 180
    3. Add 20 uL Phusion mix to 20 uL of reaction
    4. Incubate at 60C for 2 hours

Continued tomorrow