Editing
Matt:LabNotes/2014-3-26
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!
==Padlock Probe Concentration Test using Low Conc (1nM) Fixed Template== *[[Matt:LabNotes/2014-3-19 | Previously]] fixed 10uM template resulted in more targets than realistically would be in cells *24-well plate PGP1f Cells cultured w/o EGF **Fixed 3/21/14 and RT 3/21/14 **Using wells A4, A5, D2, D3, D4, D5 ***Marked 2 positions for each well (adapter clips on bottom&right) ***.maf file saved in 3-26-2014 *Test padlock probe concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 1nM, 10nM ===Fixing Template in cells=== #Pre-heat 1nM MALAT1_Template in 2X SSC to 85C (6ul 100nM MALAT1_Template + 60ul 20X SSC + 534ul H2O) #Add 100ul to each well #*Incubate @RT for 15min #Aspirate but don't wash #Add 100ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS) #*Incubate @RT for 1hr #Wash with 1X PBS twice #Add 50ul 1M Tris pH 8.0 #* Incubate @ RT for 30min #Wash with 1X PBS twice ===Anneal Padlock Probes=== *1X Ampligase Buffer *0.5 U/ul Ampligase *Incubate @ 45C for 20 hours ====Negative Control 0pM (A4)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 5U/ul Ampligase||10 |- | H2O||80 |- | Total||100 |} ====10pM (A5)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1nM ppMALAT1||1 |- | 5U/ul Ampligase||10 |- | H2O||79 |- | Total||100 |} ====50pM (D2)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1nM ppMALAT1||5 |- | 5U/ul Ampligase||10 |- | H2O||75 |- | Total||100 |} ====100pM (D3)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1nM ppMALAT1||10 |- | 5U/ul Ampligase||10 |- | H2O||70 |- | Total||100 |} ====1nM (D4)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 10nM ppMALAT1||10 |- | 5U/ul Ampligase||10 |- | H2O||70 |- | Total||100 |} ====10nM (D5)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1uM ppMALAT1||1 |- | 5U/ul Ampligase||10 |- | H2O||79 |- | Total||100 |} ===Secondary RCA=== #Aspirate but don't wash #'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. ##3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of '''2xSSC/30% formamide''' ##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) ##Add 100ul and incubate at 45C for 15min. ##Aspirate, and wash using 2x SSC twice ##Aspirate, and wash using 0.1x SSC twice #Prepare the rolling circle amplification reaction mixtures on ice. ##513 ul ddH2O, ##60 ul 10x Phi29 buffer, ##6 ul 25 mM dNTP, ##12 ul 2 mM aminoallyl dUTP ##9 ul Phi29 DNA polymerase (Epicentre, low concentration) ##*600ul Total Volume #Add 100ul each well and incubate 20hrs at 30C. (Put in at 1:30pm 3/27/14) #Wash 1X PBS once #Add 100ul BS(PEG)9 mix to each well and incubate @RT for 1hr #Wash 1X PBS twice #Add 100ul 1M Tris pH 8.0 @RT for 30min #Wash 1X PBS twice ===Detection=== *Made 600ul of 1uM dcProbe2-Cy3 (594ul 2XSSC & 30%formmaide + 6ul 100uM dcProbe2-Cy3) *Pre-heated @75C for 5 min and then added 100ul to each well *Let sit @RT for 5 min *Washed twice with 2X SSC *'''Saved in 3-28-2014''' **Imaged with 5% laser and Gain of 550 & 650 ===Results=== *Using Gain of 550 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Image''' | align="center" style="background:#f0f0f0;"|'''pp Concentration [pM]''' | align="center" style="background:#f0f0f0;"|'''Rolonies''' |- | 'Pos1_NegCtrl_Cy3_ch00_MIP.tif'||0||5963 |- | 'Pos2_NegCtrl_Cy3_ch00_MIP.tif'||0||9467 |- | 'Pos3_10pM_Cy3_ch00_MIP.tif'||10||9439 |- | 'Pos4_10pM_Cy3_ch00_MIP.tif'||10||6314 |- | 'Pos5_50pM_Cy3_ch00_MIP.tif'||50||6502 |- | 'Pos6_50pM_Cy3_ch00_MIP.tif'||50||6789 |- | 'Pos7_100pM_Cy3_ch00_MIP.tif'||100||6967 |- | 'Pos8_100pM_Cy3_ch00_MIP.tif'||100||4796 |- | 'Pos11_10nM_Cy3_ch00_MIP.tif'||10000||2167 |- | 'Pos12_10nM_Cy3_ch00_MIP.tif'||10000||6316 |} *Using Gain of 650 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Image''' | align="center" style="background:#f0f0f0;"|'''pp Concentration [pM]''' | align="center" style="background:#f0f0f0;"|'''Rolonies''' |- | 'Pos3_10pM_G650_Cy3_ch00_MIP.tif'||10||6310 |- | 'Pos4_10pM_G650_Cy3_ch00_MIP.tif'||10||6494 |- | 'Pos5_50pM_G650v2_Cy3_ch00_MIP.tif'||50||6789 |- | 'Pos6_50pM_G650_Cy3_ch00_MIP.tif'||50||6971 |- | 'Pos7_100pM_G650_Cy3_ch00_MIP.tif'||100||4796 |- | 'Pos8_100pM_G650_Cy3_ch00_MIP.tif'||100||4986 |- | 'Pos10_1nM_G650_Cy3_ch00_MIP.tif'||1000||2164 |- | 'Pos11_10nM_G650_Cy3_ch00_MIP.tif'||10000||6321 |- | 'Pos12_10nM_G650_Cy3_ch00_MIP.tif'||10000||5962 |} ===Conclusion=== *None of the sample showed significant difference from negative control so can only assume template concentration was too low *Unlike [[Matt:LabNotes/2014-3-19 | last time]] where negative control had absolutely zero rolonies, the negative control showed a LOT of '''dim''' signal **The rolonies counted here had a raw intensity value of ~1,000 **The true rolonies counted last time had a raw intensity value of ~10,000 *The other samples were also '''dim''' rolonies with intensity of ~1,000 instead of the normal ~10,000 *What can cause dim signal? **Autofluorescence ***But would expect to see this last time too unless difference between PGP1f and NCI-H1975 **Dye binding to fixed template ***Wouldn't expect to see so many since template concentration was low ***Also should've shown up in negative control last time too
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)
Template used on this page:
Template:Table
(
edit
)
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