20 % polyacrylamide gel solution was poured in to the gel assembly and comb was inserted. Gel was allowed to polymerize in at least 2 hours and assembly was pre-run in 1X TBE at 200 V for 60 minutes. The RNA samples (which were extracted earlier / without purification ) were loaded using a micro syringe and given stack run at 75 V for 15 minutes. The gel was run at 100V for 16 hours.
The gel was transferred in to a tray and RNA extract was fixed to the gel matrix in 10% trichloroacetic acid for 10 minutes on top a shaker and gel was washed twice with distilled water (DW). Gel was stained with 0.19% silver nitrate for 45 minutes on top a shaker and immediately the stained gel was washed with DW. +Viroid (Marker) bands were visualized by adding 1X NaBH4-NaOH and 2-3 drops of formaldehyde and stained gel was washed with DW. The stained gel was soaked in Na2CO3 solution to enhance bands resolution.
So, how to purify my sample RNA from Poly A gel for sequencing? What are the possible methods? I will run another PAG for excised the bands too.
I´ve isolated small fragments of transcribed RNA by passive difusion into RNA elution buffer (mainly deionized formamide, min. 4 hours - but over night is OK, and then precipitated with 1 vol. 2-propanol.
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