Hosuk:LabNotes/2013-11-7
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Padlock Probe Hybridization Test
Procedure
- Padlock probes : ACTB, RAB7A, and MALAT1
- Each concentration of Padlock probe --> 200uM in stock, use 2uM in 200uL mix
- Detection probes
- dcProbe1-Cy3 --> this does not work for padlock probe, but for 2nd Rolony detection, so we will not use this.
- Shifted FISSEQ_2ndRCAprimerDye has universal seq so it works for detection of all three padlocks
- Each step
- Step1 : ppRAB7A annealing : pre-heat @80C, add, incubate dish in oven @50C for 10min. Then add dcProbe1-Cy3, imaging
- Step2 : Strip
- Step3 : ppRAB7A+ppACTB+ppMALAT1 : pre-heat @80C, add, incubate dish in oven @50C for 30min. Then add Shifted FISSEQ_2ndRCAprimerDye, imaging
- Step4 : Strip
- Step5 : ppMALAT1 : pre-heat @80C, add, incubate dish in oven @50C for 10min. Then add Shifted FISSEQ_2ndRCAprimerDye imaging
- Step6 : add Cy5-1st Rolony detection probe without stripping
Result
- Further analysis will be done
- Pre-heating @ 80C and incubate @50C for 10 min (for 1 padlock probe) or 30min (for 3 padlock probe) worked well.
- will test lower concentration of pp.
- MALAT1 gene were many!!
- After adding Cy5-1st Rolony detection probe, MALAT1 signal became so much weaker, and number of MALAT1 lower.