Daniel:Notebook/ComboLock/2016-10-21: Difference between revisions

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>Djacobse
>Djacobse
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=Amplification Primers Test=
=Cell Test 3=


[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]


Repeating the cell test but using the qPCR primers as a metric for efficiency throughout the experiment.
==Protocol==
=Cell Trial=
<ol>
<li>C-Probe Hybridization</li>
<ol type="A">
<li>Measure cell concentrations in cell counter</li>
<li>Add cells to a new tube such that there are 100,000 cells present per sample</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt" align="center"
| align="center" width="105" height="45" | &nbsp;
|style="background-color:#CCC0DA;font-weight:bold" width="125" | C Probes-VIM (C1v2+C2)
|style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG
|- style="font-size:12pt"
| height="15"  | Sample 1
| align="center" | X
| align="center" | X
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  | Sample 2
| align="center" | X
| align="center" | X
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 3
| align="center" align="center" | &nbsp;
| align="center" | X
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 4
| align="center" | X
| align="center" align="center" | &nbsp;
|}
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li>
<li>Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O</li>
<li>Heat C probes to 90C for 5 min; Chill probes on ice to quench</li>
<li>Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample</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>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li>
</ol>
<li>Latch and Padlock Hybridization</li>
<ol type="A">
<li>Pellet at 600xg for 3 min and remove supernatant</li>
<li>Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0201-68 and 100 nM Latch0001</li>
<li>Incubate for 30 min at 37C</li>
<li>Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in 150 uL stringent wash buffer</li>
<li>Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube</li></ol>
<li>Circularization</li>
<ol type="A">
<li>Prepare 20 uL KLN mix</li>
<li>Add 2 uL KLN mix to 20 uL of reaction for sample 1</li>
<li>Incubate at 55C overnight (XX hours) </li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-10-22|tomorrow]]</li>
</ol>
=Amplification Primers Test=
Repeating the experiment from [[Daniel:Notebook/ComboLock/2016-10-20|yesterday]] with a few more conditions. Also, since that gel was saturated I'll be using lower concentrations.


==Gel Image==
==Gel Image==


[[Image:2016-10-21-AmplificationPrimers2.png|800px]]
[[Image:2016-10-21-AmplificationPrimers2.png|800px]]
[[Category:ComboLock]] [[Category:20161021]]

Revision as of 21:11, 21 October 2016

Cell Test 3

Back to Calendar

Repeating the cell test but using the qPCR primers as a metric for efficiency throughout the experiment.

Protocol

Cell Trial

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter
    2. Add cells to a new tube such that there are 100,000 cells present per sample
    3.   C Probes-VIM (C1v2+C2) U87MG
      Sample 1 X X
      Sample 2 X X
      Sample 3   X
      Sample 4 X  
    4. Pellet cells via centrifugation at 600g for 3 min
    5. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    6. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    7. Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    8. Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
    9. Pellet cells via centrifugation at 600g for 3 min
    10. Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O
    11. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    12. Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
    13. Incubate probes at 40C for 1 hour with vigorous agitation
    14. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    15. 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 with 100 nM Padlock0201-68 and 100 nM Latch0001
    3. Incubate for 30 min at 37C
    4. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
    6. Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
  3. Circularization
    1. Prepare 20 uL KLN mix
    2. Add 2 uL KLN mix to 20 uL of reaction for sample 1
    3. Incubate at 55C overnight (XX hours)
    4. Continued tomorrow


    Amplification Primers Test

    Repeating the experiment from yesterday with a few more conditions. Also, since that gel was saturated I'll be using lower concentrations.

    Gel Image

    File:2016-10-21-AmplificationPrimers2.png