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> |
Revision as of 20:41, 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 to cells at RT, and let it cool down over 5min
Annealing Universal Padlock Detection Probes
- Preheat 65C load