What are the conditions that should be taken to isolate Cccvd Viroid by Polyacrylamide gel electrophoresis?
(** CCCVd Viroids are small, single-stranded RNA molecules, ranging from 246 to 375 nucleotides long)
I've used The PAGE apparatus [ The PROTEAN® II xi cell is a large format vertical electrophoresis apparatus ] with the Voltage and Current setting given below.
20 % polyacrylamide gel solution was poured in to the gel assembly (Appendix B) 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 (see above) 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.
RNA bands visualization is in NaBH4-NaOH
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Procedure or protocol that was use in extracting the crude RNAs
Leaves were collected from frond no. 3 and wiped with moist tissue, 1 g leaflet (mid-rib free) and placed in a plastic bag with 1 volume of buffer containing 100mM Na-K Phosphate buffer, pH 7.0, 10mM EDTA and 0.5% Monothioglycerol(MTG). Leaf tissues were crushed with a hammer and squeezed-out sap into a micro centrifuge tube (approximately 300-400µl). 400µl of each volume of chloroform and aqueous phenol (90% in glass distilled water with 0.1% 8-hydroxy quinoline) was added and vortex for a few minutes. Mixture was centrifuged at 10,000 rpm for 10 min. 300µl of aqueous phase was recovered and nucleic acids with 3 volume of ethanol at -20 0C overnight. Pellet was recovered by centrifuging at 10,000rpm for 10 minutes and air dried. Pellet was dissolved in 20µl, 0.01M sodium acetate- 10% sucrose in preparation for polyacrylamide gel electrophoresis assay.
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But the bands were not expected after 16 hours.
Do the voltage and time setting have an affect on these results?? I've obtained this results 4 times repeatedly now :(
Could variation be attributed to apparatus size (distance)?
Will V,A,t setting help this?
Are Both Current and Voltage setting possible at same time ??((+_+)) ((+_+))
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