AlanFung:LabNotes/Rolony/2014-3-5: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
(8 intermediate revisions by the same user not shown) | |||
Line 3: | Line 3: | ||
*96 well plate containing NCIH-1975 cells fixed by Hosuk | *96 well plate containing NCIH-1975 cells fixed by Hosuk | ||
*Location and save location of 12 position in 96 well plate for the experiment | *Location and save location of 12 position in 96 well plate for the experiment | ||
*High Concentration(100pM) | *High Concentration (100pM) | ||
*Low Concentration (1pM) | |||
*Ultra Low Concentration (0.1pM) | |||
====Fixing Template in cells==== | ====Fixing Template in cells==== | ||
#Pre-heat 10uM MALAT1_Template in 2X SSC to 85C ( | #Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (60ul 100uM MALAT1_Template + 60ul 20X SSC + 480ul H2O) | ||
#Add 50ul to each well | #Add 50ul to each well | ||
#*Incubate @RT for 15min | #*Incubate @RT for 15min | ||
#Aspirate but don't wash | #Aspirate but don't wash | ||
#Add 50ul BS(PEG)9 mix ( | #Add 50ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS) | ||
#*Incubate @RT for 1hr | #*Incubate @RT for 1hr | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 50ul 1M Tris pH 8.0 | #Add 50ul 1M Tris pH 8.0 | ||
#* Incubate @ RT for 30min | #* Incubate @ RT for 30min | ||
====Dilute 2nM ppMALAT1==== | |||
*100pM | |||
*1pM | |||
*0.1pM | |||
====Secondary RCA==== | |||
#Aspirate each well 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 50ul to each well, 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 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14) | |||
#Wash 1X PBS once | |||
#Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS) | |||
#Wash 1X PBS twice | |||
#Add 50ul 1M Tris pH 8.0 @RT for 1hr | |||
#Wash 1X PBS twice | |||
===Anneal Padlock Probes for 24 hours=== | ===Anneal Padlock Probes for 24 hours=== | ||
#1X Ampligase Buffer | #Wash twice w PBS | ||
#Add padlock probe to each well with their corresponding concentration | #1X Ampligase Buffer (10ul of stock 10X) | ||
#0.5 U/ul Ampligase | #Add padlock probe to each well with their corresponding concentration (35ul) | ||
#0.5 U/ul Ampligase (5ul of stock 5U/ul) | |||
#Incubate @ 45C for 24 hours | #Incubate @ 45C for 24 hours | ||
#Wrapped in parafilm and put in ziploc with wet towel | #Wrapped in parafilm and put in ziploc with wet towel | ||
Line 24: | Line 54: | ||
{| 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> | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
|style="background-color:#FFFF00;font-weight:bold" width="65" colspan="3" height="15" | Group 1 | |style="background-color:#FFFF00;font-weight:bold" width="65" colspan="3" height="15" | Group 1-100pM | ||
| align="center" width="65" | | | align="center" width="65" | | ||
|style="background-color:#9BBB59;font-weight:bold" width="65" colspan="3" | Group 2 | |style="background-color:#9BBB59;font-weight:bold" width="65" colspan="3" | Group 2-1pM | ||
| align="center" width="65" | | | align="center" width="65" | | ||
|style="background-color:#4F81BD;font-weight:bold" width="65" colspan="3" | Group 3 | |style="background-color:#4F81BD;font-weight:bold" width="65" colspan="3" | Group 3-0.1pM | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
Line 49: | Line 79: | ||
| align="center" | | | align="center" | | ||
| E | | E | ||
| | | D3 | ||
| align="center" | | | align="center" | 2 | ||
| align="center" | | | align="center" | | ||
| I | | I | ||
| | | C5 | ||
| align="center" | | | align="center" | 4 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | B | | height="15" | B | ||
| | | F8 | ||
| align="center" | | | align="center" | 15 | ||
| align="center" | | | align="center" | | ||
| F | | F | ||
Line 66: | Line 96: | ||
| align="center" | | | align="center" | | ||
| J | | J | ||
| | | F6 | ||
| align="center" | | | align="center" | 7 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
Line 75: | Line 105: | ||
| align="center" | | | align="center" | | ||
| G | | G | ||
| | | C6 | ||
| align="center" | | | align="center" | 9 | ||
| align="center" | | | align="center" | | ||
| K | | K | ||
| | | C7 | ||
| align="center" | | | align="center" | 10 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | D | | height="15" | D | ||
| | | E6 | ||
| align="center" | | | align="center" | 8 | ||
| align="center" | | | align="center" | | ||
| H | | H | ||
| | | E7 | ||
| align="center" | | | align="center" | 11 | ||
| align="center" | | | align="center" | | ||
| L | | L | ||
Line 99: | Line 129: | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| align="center" width="65" height="15" | | | align="center" width="65" height="15" | | ||
| align="center" width="65" | 3 | | align="center" width="65" | 3 | ||
| align="center" width="65" | 4 | | align="center" width="65" | 4 | ||
Line 107: | Line 135: | ||
| align="center" width="65" | 7 | | align="center" width="65" | 7 | ||
| align="center" width="65" | 8 | | align="center" width="65" | 8 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | A | | height="15" | A | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
Line 129: | Line 147: | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | B | | height="15" | B | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
Line 144: | Line 156: | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | C | | height="15" | C | ||
|style="background-color:#FFFF00" | C3 | |style="background-color:#FFFF00" | C3 | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:# | |style="background-color:#4F81BD" | C5 | ||
|style="background-color:# | |style="background-color:#9BBB59" | C6 | ||
|style="background-color:#4F81BD" | C7 | |style="background-color:#4F81BD" | C7 | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | D | | height="15" | D | ||
|style="background-color:# | |style="background-color:#9BBB59" | D3 | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
Line 177: | Line 177: | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:# | |style="background-color:#FFFF00" | E6 | ||
|style="background-color:#9BBB59" | E7 | |||
|style="background-color:#9BBB59 | |||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | F | | height="15" | F | ||
|style="background-color:#FFFF00" | F3 | |style="background-color:#FFFF00" | F3 | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:#9BBB59" | F5 | |style="background-color:#9BBB59" | F5 | ||
|style="background-color:# | |style="background-color:#4F81BD" | F6 | ||
|style="background-color:#4F81BD" | F7 | |style="background-color:#4F81BD" | F7 | ||
|style="background-color:# | |style="background-color:#FFFF00" | F8 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | G | | height="15" | G | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
Line 219: | Line 201: | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | H | | height="15" | H | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | | ||
|style="background-color:#BFBFBF" align="center" | | |style="background-color:#BFBFBF" align="center" | |
Latest revision as of 00:21, 8 March 2014
Secondary Rolony Generation for Padlock Probe Concentration Test[edit]
Samples[edit]
- 96 well plate containing NCIH-1975 cells fixed by Hosuk
- Location and save location of 12 position in 96 well plate for the experiment
- High Concentration (100pM)
- Low Concentration (1pM)
- Ultra Low Concentration (0.1pM)
Fixing Template in cells[edit]
- Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (60ul 100uM MALAT1_Template + 60ul 20X SSC + 480ul H2O)
- Add 50ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Add 50ul 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
Dilute 2nM ppMALAT1[edit]
- 100pM
- 1pM
- 0.1pM
Secondary RCA[edit]
- Aspirate each well 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 50ul to each well, 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 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14)
- Wash 1X PBS once
- Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS)
- Wash 1X PBS twice
- Add 50ul 1M Tris pH 8.0 @RT for 1hr
- Wash 1X PBS twice
Anneal Padlock Probes for 24 hours[edit]
- Wash twice w PBS
- 1X Ampligase Buffer (10ul of stock 10X)
- Add padlock probe to each well with their corresponding concentration (35ul)
- 0.5 U/ul Ampligase (5ul of stock 5U/ul)
- Incubate @ 45C for 24 hours
- Wrapped in parafilm and put in ziploc with wet towel
Group 1 100pM ppMALAT1[edit]
Group 1-100pM | Group 2-1pM | Group 3-0.1pM | ||||||||
Image | Well | Mark & Find | Image | Well | Mark & Find | Image | Well | Mark & Find | ||
A | C3 | 1 | E | D3 | 2 | I | C5 | 4 | ||
B | F8 | 15 | F | F5 | 14 | J | F6 | 7 | ||
C | F3 | 13 | G | C6 | 9 | K | C7 | 10 | ||
D | E6 | 8 | H | E7 | 11 | L | F7 | 12 |
3 | 4 | 5 | 6 | 7 | 8 | |
A | ||||||
B | ||||||
C | C3 | C5 | C6 | C7 | ||
D | D3 | |||||
E | E6 | E7 | ||||
F | F3 | F5 | F6 | F7 | F8 | |
G | ||||||
H |
File:Ppconcentration NCHI1975 Group1.jpg