I DO SLOT BLOTS FOR QUANTIFICATION OF RNA FROM NEOPLASTIC CELL LINE AND DENATURE MY RNA BY FORMALDEHYDE-FORMAMIDE METHOD..
MY PROBLEM IS , SOME TIMES MY BUFFER (10X SSC ) ADDED AFTER THE APPLICATION OF THE SAMPLE RNA DOES NOT PASS THROUGH THE SLOT.
THE PROTOCOL ASKS FOR TWO WASHES OF THE SLOTS WITH 10X SSC AFTER APPLICATION OF THE SAMPLE RNA .
THE PROTOCOL ALSO ASKS FOR THE WETTING OF THE NYLON MEMBRANE WITH 20 X SSC BEFORE APPLYING THE SAMPLE.
I FOLLOW EVERY ASCEPT RELIGIOUSLY ....EVERYTHING PASSES THROUGH THE MEMBRANE .....THE WETTING BUFFER (10X SSC ),THE SAMPLE RNA ....UNTIL I DO THE LAST WASH WITH 10X SSC AGAIN.NO MATTER HOW LONG OR AT ANY PRESSURE THE BUFFER DOES NOT PASS THROUGH HENCEFORTH.
PLEASE !!!!!!!!!!!!!!!!!!!!SUGGEST.......OR CORRECT.
0
No replies to this topic
