AlanFung:LabNotes/Rolony/2013-11-14: Difference between revisions
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*In this experiment we are trying to find the concentration of probes that works best for capturing rolony targets. From previous experiments we know that a concentration of 0.6uM with a volume of 100ul works. | *In this experiment we are trying to find the concentration of probes that works best for capturing rolony targets. From previous experiments we know that a concentration of 0.6uM with a volume of 100ul works. | ||
*A test of 5 different concentrations will be perform and image analysis using PISA will be performed to count the number of target being captured | *A test of 5 different concentrations will be perform and image analysis using PISA will be performed to count the number of target being captured | ||
==MALAT1 Padlock Probes Concentrations to test == | ==MALAT1 Padlock Probes Concentrations to test == | ||
{| 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> | ||
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== | ==Setup== | ||
*In this experiment we will be using the | *In this experiment we will be using the 96well plate containing primary rolonies made on cy5 primary rolony detection probe | ||
===Reagents=== | ===Reagents=== | ||
80% Formamide in 2X SSC | 80% Formamide in 2X SSC | ||
Line 86: | Line 80: | ||
*warm up incubator to 50C for incubation | *warm up incubator to 50C for incubation | ||
*warm up tube heater to 80C for pre-heating padlock probes | *warm up tube heater to 80C for pre-heating padlock probes | ||
==Protocol== | |||
===Image and Count primary rolonies=== | |||
*Pick 6 positions and image the rolonies (Cy5) | |||
===Stripping=== | ===Stripping=== | ||
*Heat 80% formaide at 80C, add | *Heat 80% formaide at 80C, add ~50ul to 96 well plate | ||
*Incubate for 15 mins wash 3 times with | *Incubate for 15 mins wash 3 times with 200ul pbs | ||
===Annealing Padlock Probes=== | ===Annealing Padlock Probes=== | ||
*Pre-heat padlock probes at 80C for 5 mins | *Pre-heat padlock probes at 80C for 5 mins | ||
*Add to cells at RT | *aspirate | ||
*Add 50ul to cells at RT | |||
*transfer to 50C for 15 mins | |||
*aspirate and wash with pbs 3x | |||
===Annealing Universal Padlock Detection Probes=== | ===Annealing Universal Padlock Detection Probes=== | ||
*Preheat 65C | *Preheat detection probes at 65C for 5 mins (2uM) | ||
*Add 100ul to cells at RT incubate for 15 mins | |||
*Wash with H2O twice and add 100ul 2x SSC | |||
===Imaging=== | ===Imaging=== | ||
*10-15um, 10 z stack image, lower limit begin go up till upper limit end | |||
*resolution (2kx2k) | |||
*16bit,600Hz | |||
*when done save file (experiment_dish(mattekdate)_step#_dyeprobe_channel) | |||
==Protocol for stripping efficiency test== |
Latest revision as of 19:45, 21 November 2013
Optimum Probe# Test[edit]
- In this experiment we are trying to find the concentration of probes that works best for capturing rolony targets. From previous experiments we know that a concentration of 0.6uM with a volume of 100ul works.
- A test of 5 different concentrations will be perform and image analysis using PISA will be performed to count the number of target being captured
MALAT1 Padlock Probes Concentrations to test[edit]
5uM |
100nM |
10nM |
1nM |
0.1nM |
Setup[edit]
- In this experiment we will be using the 96well plate containing primary rolonies made on cy5 primary rolony detection probe
Reagents[edit]
80% Formamide in 2X SSC
100ul MALAT1 padlock probes in 2X ssc in various concentration (ppMALAT1)
- Original concentration 100uM, perform serial dilution
Stock Tube | Stock Volume | 2XSSC | Dilution | Final concentration nM |
100000 | 7.5 | 142.5 | 0.05 | 5000 |
5000 | 3 | 147 | 0.02 | 100 |
100 | 15 | 135 | 0.1 | 10 |
10 | 15 | 135 | 0.1 | 1 |
1 | 15 | 135 | 0.1 | 0.1 |
MALAT1 padlock probes detection
- Dilute 110ul 10uM DC_Probe_2_Cy3 with 440ul 2X SSC
- Shifted FISSEQ_2ndRCAprimerDye : 2nd Rolony from all padlock probes GGATATCGGGAAGCTGAA/3ATTO488N/
Apparatus[edit]
- warm up incubator to 50C for incubation
- warm up tube heater to 80C for pre-heating padlock probes
Protocol[edit]
Image and Count primary rolonies[edit]
- Pick 6 positions and image the rolonies (Cy5)
Stripping[edit]
- Heat 80% formaide at 80C, add ~50ul to 96 well plate
- Incubate for 15 mins wash 3 times with 200ul pbs
Annealing Padlock Probes[edit]
- Pre-heat padlock probes at 80C for 5 mins
- aspirate
- Add 50ul to cells at RT
- transfer to 50C for 15 mins
- aspirate and wash with pbs 3x
Annealing Universal Padlock Detection Probes[edit]
- Preheat detection probes at 65C for 5 mins (2uM)
- Add 100ul to cells at RT incubate for 15 mins
- Wash with H2O twice and add 100ul 2x SSC
Imaging[edit]
- 10-15um, 10 z stack image, lower limit begin go up till upper limit end
- resolution (2kx2k)
- 16bit,600Hz
- when done save file (experiment_dish(mattekdate)_step#_dyeprobe_channel)