Northern and Southern Blotting, and Colony and Plaque Lifts

Southern Blotting

Direct comparison of three charged nylon membranes in their own optimized protocols: Each membrane was cross-linked at its recommended UV wavelength. Using these cross-linking conditions, Immobilon-Ny+ membrane had twice the hybridization signal of both competitors.

Signal Intensity after 12 Reprobes

Nylon membranes were cross-linked at 254 nm and underwent multiple reprobes including, in sequence, two hybridizations with ³²P-labeled probe, four mock hybridizations, a seventh hybridization with ³²P-labeled probe followed by five mock hybridizations and a thirteenth hybridization with ³²P-labeled probe. The membranes were stripped in 0.4 M NaOH at 45 °C for 30 minutes between each cycle of hybridization. The mock hybridizations were the same as the true hybridizations except that no ³²P-labeled probe was added. Throughout the reprobing process, Immobilon-Ny+ performed better with twice the signal than both Competitor A and B's charged nylon membranes.

Detection Sensitivity

Amount of DNA loaded on the gel was decreased to 0.01 ng. The 2.2 and 2.0 kbp bands in the 0.1 lane correspond to 0.48 and 0.42 pg of DNA, respectively. This is approximately equivalent to a single copy gene sequence in 10 µg of human genomic DNA. The sensitivity was improved by doubling the probe concentration. In the 0.01 ng lanes, Immobilon-Ny+ membrane was slightly more sensitive with lower background than Competitor A.

Northern Blotting with Chemiluminescent Detection

Mouse liver total RNA (1 µg) was resolved on a formaldehyde agarose gel and transferred to Immobilon-Ny+ and a competitor's positively charged nylon membrane "A" by capillary blotting. Fixation of the RNA by UV cross-linking enhanced the signal intensity when compared to baking. Immobilon-Ny+ membrane shows better sensitivity than Competitor A.

Colony Lifts

A mixed bacterial suspension of JM109 carrying pUC19 or pLH2 (2.0 kbp fragment of lambda phage DNA Hind III cloned into pUC19) was plated on LB agar plates and incubated at 37 °C overnight. After chilling the plates at 4 °C for 30 minutes, the colonies (approximately 1–2 mm diameter) were transferred to an 82 mm disc membrane of Immobilon-Ny+ or a competitor's positively charged nylon. The DNA on the membrane was UV cross-linked with 60,000 µJoules/cm² at 254 nm. The membranes were then hybridized with a ³²P labeled DNA probe and visualized using a phosphor imaging system.