Daniel:Notebook/ComboLock/2016-6-23: Difference between revisions

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==Protocol==
==Protocol==


===Prep===
'''Wash Buffer'''
{| 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="110" height="42" | Reagent
| width="65" | Stock
| width="65" | Final
| width="65" | Dilution
| width="82" | Amt in 10 mL
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | PBS
| align="center" valign="bottom" | 10X
| align="center" valign="bottom" | 1X
| align="center" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 1 mL
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Tween 20
| align="center" align="center" valign="bottom" | 100%
| align="center" align="center" valign="bottom" | 0.10%
| align="center" align="center" valign="bottom" | 1000
| align="center" valign="bottom" | 10 uL
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 4 U/mL
| align="center" align="center" valign="bottom" | 1000
| align="center" valign="bottom" | 10 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" valign="bottom" | 9 mL
|}
'''Stringent Wash Buffer'''
{| 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="110" height="42" | Reagent
| width="65" | Stock
| width="65" | Final
| width="65" | Dilution
| width="74" | Amt in 1 mL (uL)
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | PBS
| align="center" valign="bottom" | 10X
| align="center" valign="bottom" | 1X
| align="center" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 100 uL
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | SSC
| align="center" valign="bottom" | 20X
| align="center" valign="bottom" | 4X
| align="center" align="center" valign="bottom" | 5
| align="center" valign="bottom" | 200 uL
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" align="center" valign="bottom" | 100
| align="center" valign="bottom" | 10 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" valign="bottom" | 690 uL
|}
'''LPH Buffer'''
{| 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="110" height="42" | Reagent
| width="65" | Stock
| width="65" | Final
| width="65" | Dilution
| width="74" | Amt in 100 uL (uL)
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Oligos
| align="center" valign="bottom" | 10 uM
| align="center" valign="bottom" | 100 nM
| align="center" align="center" valign="bottom" | 100
| align="center" valign="bottom" | 1 uL
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | SSC
| align="center" valign="bottom" | 20X
| align="center" valign="bottom" | 1X
| align="center" align="center" valign="bottom" | 5
| align="center" valign="bottom" | 20 uL
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" align="center" valign="bottom" | 100
| align="center" valign="bottom" | 1 uL
|- style="background-color:#D9D9D9;font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | PBS
| align="center" valign="bottom" | 10X
| align="center" valign="bottom" | 1X
| align="center" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 10 uL
|- style="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" valign="bottom" | 68 uL
|}
===Experiment===
'''Sample Matrix'''
'''Sample Matrix'''
{| 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 31: Line 161:
<li>Split cells into 2 samples (1 mL methanol each)</li>
<li>Split cells into 2 samples (1 mL methanol each)</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Put 1 uL C1 Probe and 1 uL C2 probe into a 0.2 mL tube</li>
<li>Combine 1.5 uL of each C1 probe into a 0.2 mL tube</li>
<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 probes to CP buffer for final concentration of 100 nM (Add 1 uL probe mixture to 0.5mL hybridization buffer)</li>
<li>Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Incubate probes at 40C for 1 hour with vigorous agitation</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
<li>Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min</li>
Line 41: Line 171:
<ol type="A">
<ol type="A">
<li>Pellet at 600xg for 3 min and remove supernatant</li>
<li>Pellet at 600xg for 3 min and remove supernatant</li>
<li>Resuspend pellet in 50 uL hybridization buffer with 100 nM insert/backbone oligos for 30 min at 37C</li>
<li>Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)</li>
<li>Wash twice</li>
<li> Add 1 uL of each insert/backbone (final conc. 100 nM each)</li>
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol></ol>
 
'''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="65" height="30" | Batch
| width="65" | Cell Sample
| width="65" | Latch0001 (1 uL)
| width="65" | Latch0002 (1 uL)
| width="89" | Padlock0001-48 (1 uL)
| width="87" | Padlock0001-68 (1 uL)
| width="91" | Padlock0001-88 (1 uL)
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Batch 1
| align="center" | S1
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Batch 2
| align="center" | S1
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" | &nbsp;
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Batch 3
| align="center" | S1
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
| align="center" | X
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Batch 4
| align="center" | S1
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Batch 5
| align="center" | S1
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" | &nbsp;
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Batch 6
| align="center" | S1
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
| align="center" | X
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Batch 7
| align="center" | S2
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Batch 8
| align="center" | S2
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
 
|}
<li>Incubate for 30 min at 37C following the sample matrix</li>
<li>Wash twice with 200 uL wash buffer</li>
<ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol>
<li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol>
<li>Circularization</li>
<li>Circularization</li>
<ol type="A">
<ol type="A">
<li>Prepare KLN mix with:</li>
<li>Prepare 20 uL KLN mix</li>
<ol type="a">
<li>20% v/v HemoKlentaq</li>
<li>0.5 U/μL Ampligase</li>
<li>100 μM of dNTP mix</li>
<li>1x Ampligase Buffer</li>
</ol>
<li>Add 2 uL KLN mix to 20 uL of reaction</li>
<li>Add 2 uL KLN mix to 20 uL of reaction</li>
<li>Incubate at 55C for 4-20 hours</li>
<li>Incubate at 55C overnight (XX hours) </li>
<li>Heat inactivate enzyme by incubating for 2 minutes at 94C</li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-6-24|tomorrow]]</li>
</ol>
<li>Exonuclease Digestion</li>
<ol type="A">
<li>Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio</li>
<li>Add 2 μL of exonuclease I/III mix to reaction</li>
<li>Mix the reaction by swirling pipette around the well 5 times</li>
<li>Incubate reaction at 37 ºC for 2 hours</li>
<li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li>
</ol>
<li>qPCR</li>
<ol type="A">
<li>Set up reaction according to table</li>
<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 x20</li>
<li>72C 2 min</li>
<li>16C hold</li>
</ol>
</ol>
</ol>
</ol>


Line 85: Line 274:


<ol type="A">
<ol type="A">
<li>C-Probe (CP) Buffer</li>
<li>C-Probe Hybridization (CPH) Buffer</li>
<ol type="a">
<ol type="a">
<li>100 nM insert/backbone oligos</li>
<li>100 nM insert/backbone oligos</li>
Line 98: Line 287:
<li>4 U/mL RNasin</li>
<li>4 U/mL RNasin</li>
</ol>
</ol>
<li>Latch-Padlock Hybridization (LPH) Buffer</li>
<ol type="a">
<li>100 nM insert/backbone oligos</li>
<li>1X SSC</li>
<li>40 U/mL RNasin</li>
<li>PBS</li>
</ol>
<li>KLN Mix 20 uL total
<ol type="a">
<li> 4 uL HemoKlentaq (20% v/v)</li>
<li> 2 μL Ampligase (0.5 U/uL)</li>
<li> 2 uL 1 mM dNTP mix (100 μM ea)</li>
<ol type="i"><li>Add 10 uL 10mM dNTP mix to 90 uL nfH2O</li></ol>
<li> 2 uL 1x Ampligase Buffer 1x</li>
<li> 10 uL nfH2O</li>
</ol>
[[Category:ComboLock]] [[Category:20160623]]

Latest revision as of 22:30, 6 July 2016

Stage 2 Test[edit]

Back to Calendar

The purpose of this experiment is to test the latch and padlock binding, termed stage 2 (at the moment). This is based on the success of the VIM binding for both C-Probes and antibodies.

Protocol[edit]

Prep[edit]

Wash Buffer

Reagent Stock Final Dilution Amt in 10 mL
PBS 10X 1X 10 1 mL
Tween 20 100% 0.10% 1000 10 uL
Rnasin 40000 U/mL 4 U/mL 1000 10 uL
nf H2O NA NA NA 9 mL

Stringent Wash Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
PBS 10X 1X 10 100 uL
SSC 20X 4X 5 200 uL
Rnasin 40000 U/mL 40 U/mL 100 10 uL
nf H2O NA NA NA 690 uL

LPH Buffer

Reagent Stock Final Dilution Amt in 100 uL (uL)
Oligos 10 uM 100 nM 100 1 uL
SSC 20X 1X 5 20 uL
Rnasin 40000 U/mL 40 U/mL 100 1 uL
PBS 10X 1X 10 10 uL
nf H2O NA NA NA 68 uL

Experiment[edit]

Sample Matrix

Sample VIM-C1 (1 uL) VIM-C2 (1 uL)
Sample 1 X X
Sample 2    
  1. C-Probe Hybridization
    1. Split cells into 2 samples (1 mL methanol each)
    2. Pellet cells via centrifugation at 600g for 3 min
    3. Combine 1.5 uL of each C1 probe into a 0.2 mL tube
    4. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    5. Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)
    6. Incubate probes at 40C for 1 hour with vigorous agitation
    7. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    8. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
  2. Latch and Padlock Hybridization
    1. Pellet at 600xg for 3 min and remove supernatant
    2. Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)
    3. Add 1 uL of each insert/backbone (final conc. 100 nM each)
    4. Sample Matrix
      Batch Cell Sample Latch0001 (1 uL) Latch0002 (1 uL) Padlock0001-48 (1 uL) Padlock0001-68 (1 uL) Padlock0001-88 (1 uL)
      Batch 1 S1 X   X    
      Batch 2 S1 X     X  
      Batch 3 S1 X       X
      Batch 4 S1   X X    
      Batch 5 S1   X   X  
      Batch 6 S1   X     X
      Batch 7 S2 X   X    
      Batch 8 S2   X X    
    5. Incubate for 30 min at 37C following the sample matrix
    6. Wash twice with 200 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    7. Resuspend with 20 uL 1x Amp Ligase buffer
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow

    Buffers[edit]

    1. C-Probe Hybridization (CPH) Buffer
      1. 100 nM insert/backbone oligos
      2. 1X SSC
      3. 40 U/mL RNasin
      4. PBS
    2. Wash Buffer
      1. PBS
      2. 0.1% Tween
      3. 4 U/mL RNasin
    3. Latch-Padlock Hybridization (LPH) Buffer
      1. 100 nM insert/backbone oligos
      2. 1X SSC
      3. 40 U/mL RNasin
      4. PBS
    4. KLN Mix 20 uL total
      1. 4 uL HemoKlentaq (20% v/v)
      2. 2 μL Ampligase (0.5 U/uL)
      3. 2 uL 1 mM dNTP mix (100 μM ea)
        1. Add 10 uL 10mM dNTP mix to 90 uL nfH2O
      4. 2 uL 1x Ampligase Buffer 1x
      5. 10 uL nfH2O