AlanFung:LabNotes/Rolony/2013-11-14: Difference between revisions
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*Pre-heat padlock probes at 80C for 5 mins | *Pre-heat padlock probes at 80C for 5 mins | ||
*Add 100ul to cells at RT, and let it cool down over 5min | *Add 100ul to cells at RT, and let it cool down over 5min | ||
*transfer to 50C for | |||
*aspirate and wash with pbs 3x | *aspirate and wash with pbs 3x | ||
Revision as of 21:02, 19 November 2013
Optimum Probe# Test
- 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
5uM |
100nM |
10nM |
1nM |
0.1nM |
Protocol
- In this experiment we will be using the Mattek dish containing primary rolonies made on Oct 23rd dish #2
Reagents
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
- warm up incubator to 50C for incubation
- warm up tube heater to 80C for pre-heating padlock probes
Stripping
- Heat 80% formaide at 80C, add 200ul to mattek dish
- Incubate for 15 mins wash 3 times with 200ul pbs
Annealing Padlock Probes
- Pre-heat padlock probes at 80C for 5 mins
- Add 100ul to cells at RT, and let it cool down over 5min
- transfer to 50C for
- aspirate and wash with pbs 3x
Annealing Universal Padlock Detection Probes
- Preheat detection probes at 65C for 5 mins
- Add 100ul to cells at RT incubate for
- Wash with H2O twice and add 100ul 2x SSC
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)