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*[[Hosuk:Lab_Notes|LabNote]] ===Padlock Probe Hybridization Test=== ====Procedure==== *Padlock probes : ACTB, RAB7A, and MALAT1 **Each concentration of Padlock probe were **200uM in stock, use 2uM in 200uL mix when adding a single padlock probe **For 3 padlock probes, we mixed 67uL from each padlock probes to make 200uL, so each padlock probe concentration became 0.67uM *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 ο 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.
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