Editing
Daniel:Notebook/ComboLock/2016-7-22
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=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. ==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== <ol> <li>C-Probe Hybridization</li> <ol type="A"> <li>Measure cell concentrations in cell counter (Human - 400,000 cells/mL)</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>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> <li>Resuspend cells in 100 uL CPH buffer</li> <li>Incubate probes at 40C for 1 hour with vigorous agitation</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 150 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</li> <li>Incubate for 30 min at 37C following the sample matrix</li> <li>Wash twice with 100 uL wash buffer</li> <ol type="a"><li>Wash by pelleting cells at 600xg for 3 min</li></ol> <li>Resuspend pellet in 100 uL LPH buffer</li> <li>Incubate for 30 min at 37C following the sample matrix</li> <li>Wash twice with 100 uL wash buffer pelleting cells at 1000xg for 3 min</li> <li>Resuspend with 20 uL 1x Amp Ligase buffer</li></ol> </ol> 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" | | align="center" align="center" valign="bottom" | |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" | |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" | | 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" | | 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" | | align="center" | 10X Latch |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | Sample 4 | align="center" align="center" | | align="center" align="center" | | align="center" | X | align="center" align="center" | | 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" | | 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" | | align="center" align="center" valign="bottom" | | 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:20160722A]] [[Category:20160722B]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information