Daniel:Notebook/ComboLock/2016-7-22: Difference between revisions

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>Djacobse
(Created page with "=Cell Retention Test= I've been worried about cell retention during the spins for some time, and I've been thinking about how to test it. So, I'm going to run through the pro...")
 
>Djacobse
 
(22 intermediate revisions by the same user not shown)
Line 1: Line 1:
=Cell Retention Test=
=Cell Retention Test=
[[Daniel:Notebook/ComboLock|Back to Calendar]]


I've been worried about cell retention during the spins for some time, and I've been thinking about how to test it. So, I'm going to run through the protocol (without probes) but using hematoxylin to stain the cells.  
I've been worried about cell retention during the spins for some time, and I've been thinking about how to test it. So, I'm going to run through the protocol (without probes) but using hematoxylin to stain the cells.  
==Buffers==
'''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" | 1000
| align="center" valign="bottom" | 1 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" | 700 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="65" height="30" | Reagent
| width="105" | Stock
| width="65" | Final
| width="65" | Dilution
| width="65" | Amt in 1 mL (uL)
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Oligos
| align="center" valign="bottom" | 100 uM
| align="center" valign="bottom" | 100 nM
| align="center" align="center" valign="bottom" | 1000
| 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" | 1X
| align="center" align="center" valign="bottom" | 20
| align="center" valign="bottom" | 50 uL
|- style="font-size:12pt"
|style="font-weight:bold" height="30"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" align="center" valign="bottom" | 1000
| 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" | 100 uL
|- style="font-size:12pt"
|style="font-weight:bold" height="30"  valign="bottom" | nf H2O
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 850 uL
|}


==Protocol==
==Protocol==
Line 8: Line 99:
<li>C-Probe Hybridization</li>
<li>C-Probe Hybridization</li>
<ol type="A">
<ol type="A">
<li>Measure cell concentrations in cell counter (Mouse - cells/mL)</li>
<li>Measure cell concentrations in cell counter (Human - 400,000 cells/mL)</li>
<li>Add cells to a new tube such that there are 100000 cells present ( uL)</li>
<li>Add cells to a new tube such that there are 200,000 cells present (500 uL)</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash with 200 uL PBS and 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>Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash 3 times with 200 uL PBS and pellet cells via centrifugation at 600g for 3 min</li>
<li>Wash 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li>
<li>Resuspend cells in 100 uL CPH buffer</li>
<li>Resuspend cells in 100 uL CPH 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 100 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>
<li>Incubate cells for 20 min at 40C in 150 uL stringent wash buffer</li>
</ol>
</ol>
<li>Latch and Padlock Hybridization</li>
<li>Latch and Padlock Hybridization</li>
Line 32: Line 123:
</ol>
</ol>


This is the last step where you change buffer in the protocol. If I still have cells, we're good.
This is the last step where you change buffer in the protocol. If I still have cells, we're good.  
 
==Results==
 
I still had cells at this point, but the hematoxylin really helps visualize the cell pellet and doesn't interfere with the chemistry (so it has been reported) so I think that it is a good idea to use it.
 
{| 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="59" height="30" | Sample
| width="65" | Cells/mL beginning
| width="65" | mL start
| width="65" | Total Cells Beginning
| width="65" | Cells/mL End
| width="65" | mL End
| width="65" | Total Cells End
| width="65" | Yield (%)
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | CRT-A
| align="center" | 1.06E+06
| align="center" | 0.5
| align="center" | 5.30E+05
| align="center" | 1.38E+06
| align="center" | 1.00E-01
| align="center" | 1.38E+05
| align="center" | 10.0%
 
|- style="background-color:#D9D9D9;font-size:12pt" align="center" valign="bottom"
| height="15" | CRT-B
| align="center" | 9.35E+05
| align="center" | 0.5
| align="center" | 4.68E+05
| align="center" | 1.63E+06
| align="center" | 1.00E-01
| align="center" | 1.63E+05
| align="center" | 34.9%
 
|}
 
=C Probe Test 3-SOD1/VIM-Stringent Washes (Started [[Daniel:Notebook/ComboLock/2016-7-18|7-18-2016]])=
 
<ol start="6">
<li>Ethanol precipitation (from [[Daniel:Notebook/ComboLock/2016-7-21|yesterday]])</li>
<ol type="A" start="3">
<li>Centrifuge at 4 C for 25 minutes at 14000 rpm
<li>Remove supernatant and add 750 uL chilled 75% EtOH</li>
<li>Centrifuge for 15 minutes at minutes at 4 C at 14000 rpm</li>
<li>Remove supernatant and dry the pellet in the hood</li>
<li>Resuspend pellet in 20 uL volume nfH20</li>
<li>Measure in Nanodrop</li>
<li>Combined 15 uL from each sample</li>
<li>Store DNA at 4C</li>
</ol></ol>
 
===Nanodrop Results===
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#DDD9C4;font-size:12pt;font-weight:bold" align="center"
| width="185" height="15" | Source
| width="65" | ng/uL
 
|- style="font-size:12pt"
| height="30"  | 7-21 (Stringent Wash) A
| align="center" align="center" | 44.5
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="30"  | 7-21 (Stringent Wash) B
| align="center" align="center" | 13.9
 
|}
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#DDD9C4;font-size:12pt;font-weight:bold" align="center"
| width="106" height="30" | Sample
| width="65" | ng/uL
| width="65" | uL added
| width="65" | final mass (ng)
 
|- style="font-size:12pt"
| height="15"  | 7-21 Sample A
| align="center" align="center" | 44.5
| align="center" align="center" | 15
| align="center" align="center" | 667.5
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  | 7-21 Sample B
| align="center" align="center" | 13.9
| align="center" align="center" valign="bottom" | 15
| align="center" align="center" valign="bottom" | 208.5
 
|}
 
'''20160722_DEJ_CL_C04'''
 
=Latch 100 and Dextran Test=
 
==Buffer Prep==
 
'''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="65" height="30" | Reagent
| width="65" | Stock
| width="65" | Final
| width="65" | Dilution
| width="65" | Amt in 5 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" | 500 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" | 1000 uL
 
|- style="font-size:12pt"
|style="font-weight:bold" height="30"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" align="center" valign="bottom" | 1000
| align="center" valign="bottom" | 5 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" | 4.5 mL
 
|}
 
'''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="65" height="30" | Reagent
| width="105" | Stock
| width="65" | Final
| width="65" | Dilution
| width="65" | Amt in 1 mL (uL)
 
|- style="font-size:12pt"
|style="font-weight:bold" height="15"  valign="bottom" | Oligos
| align="center" valign="bottom" | 100 uM
| align="center" valign="bottom" | 100 nM
| align="center" align="center" valign="bottom" | 1000
| 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" | 1X
| align="center" align="center" valign="bottom" | 20
| align="center" valign="bottom" | 50 uL
 
|- style="font-size:12pt"
|style="font-weight:bold" height="30"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" align="center" valign="bottom" | 1000
| 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" | 100 uL
 
|- style="font-size:12pt"
|style="font-weight:bold" height="30"  valign="bottom" | nf H2O
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 850 uL
 
|}
 
'''CPH Buffer with 10% Dextran Sulfate'''
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#D8E4BC;font-size:12pt;font-weight:bold" align="center"
| width="165" height="30" | Reagent
| width="105" | Stock Conc.
| width="105" | Final Conc.
| width="65" | Amt. Added
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | SSC
| align="center" valign="bottom" | 20X
| align="center" valign="bottom" | 1X
| align="center" valign="bottom" | 50 uL
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Tween 20
| align="center" align="center" valign="bottom" | 10%
| align="center" align="center" valign="bottom" | 0.10%
| align="center" valign="bottom" | 10 uL
 
|- style="font-size:12pt"
| height="21"  valign="bottom" | Rnasin
| align="center" valign="bottom" | 40000 U/mL
| align="center" valign="bottom" | 40 U/mL
| align="center" valign="bottom" | 1 uL
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="30"  valign="bottom" | Riboside Vanadyl Complex
| align="center" valign="bottom" | 200 mM
| align="center" valign="bottom" | 20 mM
| align="center" valign="bottom" | 100 uL
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Poly Vinylsulfonic Acid
| align="center" align="center" valign="bottom" | 25%
| align="center" align="center" valign="bottom" | 2.50%
| align="center" valign="bottom" | 100 uL
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Salmon Sperm
| align="center" valign="bottom" | 10 mg/mL
| align="center" valign="bottom" | 100 ug/mL
| align="center" valign="bottom" | 10 uL
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Dextran Sulfate
| align="center" align="center" valign="bottom" | 25%
| align="center" align="center" valign="bottom" | 10%
| align="center" valign="bottom" | 400 uL
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Probes
| align="center" valign="bottom" | 100 uM
| align="center" valign="bottom" | 100 nM
| align="center" valign="bottom" | 1 uL/probe
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Water
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | NA
| align="center" valign="bottom" | 340 uL
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Total
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
|style="font-weight:bold" align="center" valign="bottom" | 1 mL
 
|}
 
==Protocol==
 
<ol>
<li>C-Probe Hybridization</li>
<ol type="A">
<li>Measure cell concentrations in cell counter</li>
<ol type="a"><li>Human - cells/mL; Mouse - cells/mL</li></ol>
<li>Add cells to a new tube such that there are 100000 cells present</li>
<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 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min</li>
<ol type="a"><li>Human- uL; Mouse- uL</li></ol>
<li>Pellet cells via centrifugation at 600g for 3 min</li>
<li>Combine 17 uL of each C 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>Add probes to CPH buffer according to sample matrix</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="65" | C Probes-VIM (C1+C2)
|style="background-color:#CCC0DA;font-weight:bold" width="65" | C Probes-SOD1 (C1+C2)
|style="background-color:#CCC0DA;font-weight:bold" width="65" | U87MG
|style="background-color:#CCC0DA;font-weight:bold" width="65" | 3T3 Cells
|style="background-color:#CCC0DA;font-weight:bold" width="65" | Treatment
 
|- style="font-size:12pt"
| height="15"  | Sample 1
| align="center" | X
| align="center" | X
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" | None
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  | Sample 2
| align="center" | X
| align="center" | X
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" | Dextran
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 3
| align="center" | X
| align="center" | X
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" | 10X Latch
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 4
| align="center" align="center" | &nbsp;
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" valign="bottom" | None
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 5
| align="center" | X
| align="center" | X
| align="center" align="center" | &nbsp;
| align="center" | X
| align="center" | None
 
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 6
| align="center" valign="bottom" | X
| align="center" valign="bottom" | X
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | &nbsp;
| align="center" | None
 
|}
<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 Padlock0001-68 </li>
<li>Incubate for 30 min at 37C</li>
<li>Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li>
<li>Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001</li>
<li>Incubate for 30 min at 37C</li>
<li>Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min</li>
<li>Incubate cells for 20 min at 40C in 200 uL stringent wash buffer</li>
<li>Resuspend with 20 uL 1x Amp Ligase buffer</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</li>
<li>Incubate at 55C overnight (XX hours) </li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-7-23|tomorrow]]</li>
</ol>
 


[[Category:ComboLock]] [[Category:20160722]]
[[Category:ComboLock]] [[Category:20160722A]] [[Category:20160722B]]

Latest revision as of 19:04, 28 July 2016

Cell Retention Test[edit]

Back to Calendar

I've been worried about cell retention during the spins for some time, and I've been thinking about how to test it. So, I'm going to run through the protocol (without probes) but using hematoxylin to stain the cells.

Buffers[edit]

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 1000 1 uL
nf H2O NA NA NA 700 uL

LPH Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
Oligos 100 uM 100 nM 1000 100 uL
SSC 20X 1X 20 50 uL
Rnasin 40000 U/mL 40 U/mL 1000 1 uL
PBS 10X 1X 10 100 uL
nf H2O NA NA NA 850 uL

Protocol[edit]

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter (Human - 400,000 cells/mL)
    2. Add cells to a new tube such that there are 200,000 cells present (500 uL)
    3. Pellet cells via centrifugation at 600g for 3 min
    4. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    5. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    6. Wash 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    7. Resuspend cells in 100 uL CPH buffer
    8. Incubate probes at 40C for 1 hour with vigorous agitation
    9. Wash three times with 100 uL wash buffer and pelleting at 600xg for 3 min
    10. Incubate cells for 20 min at 40C in 150 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
    3. Incubate for 30 min at 37C following the sample matrix
    4. Wash twice with 100 uL wash buffer
      1. Wash by pelleting cells at 600xg for 3 min
    5. Resuspend pellet in 100 uL LPH buffer
    6. Incubate for 30 min at 37C following the sample matrix
    7. Wash twice with 100 uL wash buffer pelleting cells at 1000xg for 3 min
    8. Resuspend with 20 uL 1x Amp Ligase buffer

This is the last step where you change buffer in the protocol. If I still have cells, we're good.

Results[edit]

I still had cells at this point, but the hematoxylin really helps visualize the cell pellet and doesn't interfere with the chemistry (so it has been reported) so I think that it is a good idea to use it.

Sample Cells/mL beginning mL start Total Cells Beginning Cells/mL End mL End Total Cells End Yield (%)
CRT-A 1.06E+06 0.5 5.30E+05 1.38E+06 1.00E-01 1.38E+05 10.0%
CRT-B 9.35E+05 0.5 4.68E+05 1.63E+06 1.00E-01 1.63E+05 34.9%

C Probe Test 3-SOD1/VIM-Stringent Washes (Started 7-18-2016)[edit]

  1. Ethanol precipitation (from yesterday)
    1. Centrifuge at 4 C for 25 minutes at 14000 rpm
    2. Remove supernatant and add 750 uL chilled 75% EtOH
    3. Centrifuge for 15 minutes at minutes at 4 C at 14000 rpm
    4. Remove supernatant and dry the pellet in the hood
    5. Resuspend pellet in 20 uL volume nfH20
    6. Measure in Nanodrop
    7. Combined 15 uL from each sample
    8. Store DNA at 4C

Nanodrop Results[edit]

Source ng/uL
7-21 (Stringent Wash) A 44.5
7-21 (Stringent Wash) B 13.9
Sample ng/uL uL added final mass (ng)
7-21 Sample A 44.5 15 667.5
7-21 Sample B 13.9 15 208.5

20160722_DEJ_CL_C04

Latch 100 and Dextran Test[edit]

Buffer Prep[edit]

Stringent Wash Buffer

Reagent Stock Final Dilution Amt in 5 mL (uL)
PBS 10X 1X 10 500 uL
SSC 20X 4X 5 1000 uL
Rnasin 40000 U/mL 40 U/mL 1000 5 uL
nf H2O NA NA NA 4.5 mL

LPH Buffer

Reagent Stock Final Dilution Amt in 1 mL (uL)
Oligos 100 uM 100 nM 1000 100 uL
SSC 20X 1X 20 50 uL
Rnasin 40000 U/mL 40 U/mL 1000 1 uL
PBS 10X 1X 10 100 uL
nf H2O NA NA NA 850 uL

CPH Buffer with 10% Dextran Sulfate

Reagent Stock Conc. Final Conc. Amt. Added
SSC 20X 1X 50 uL
Tween 20 10% 0.10% 10 uL
Rnasin 40000 U/mL 40 U/mL 1 uL
Riboside Vanadyl Complex 200 mM 20 mM 100 uL
Poly Vinylsulfonic Acid 25% 2.50% 100 uL
Salmon Sperm 10 mg/mL 100 ug/mL 10 uL
Dextran Sulfate 25% 10% 400 uL
Probes 100 uM 100 nM 1 uL/probe
Water NA NA 340 uL
Total     1 mL

Protocol[edit]

  1. C-Probe Hybridization
    1. Measure cell concentrations in cell counter
      1. Human - cells/mL; Mouse - cells/mL
    2. Add cells to a new tube such that there are 100000 cells present
    3. Pellet cells via centrifugation at 600g for 3 min
    4. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    5. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    6. Wash 3 times with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
      1. Human- uL; Mouse- uL
    7. Pellet cells via centrifugation at 600g for 3 min
    8. Combine 17 uL of each C probe into a 0.2 mL tube
    9. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    10. Add probes to CPH buffer according to sample matrix
    11.   C Probes-VIM (C1+C2) C Probes-SOD1 (C1+C2) U87MG 3T3 Cells Treatment
      Sample 1 X X X   None
      Sample 2 X X X   Dextran
      Sample 3 X X X   10X Latch
      Sample 4     X   None
      Sample 5 X X   X None
      Sample 6 X X     None
    12. Incubate probes at 40C for 1 hour with vigorous agitation
    13. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    14. 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 Padlock0001-68
    3. Incubate for 30 min at 37C
    4. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    6. Resuspend pellet in 100 uL LPH buffer with 100 nM Latch0001
    7. Incubate for 30 min at 37C
    8. Wash twice with 200 uL wash buffer by pelleting cells at 600xg for 3 min
    9. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
    10. 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