Northern Blotting (use by DIG)

목차

Additional Reagents Required

Procedure

Digoxigenin-labeled probe DNA preparation with PCR

본문내용

Procedure

1. Agarose gel preparation
․ Melt 1g of agarose in a solution made with the following.
10㎖ of 10x MOPS buffer
85㎖ of sterile water
․ Allow the melted agarose solution to cool to ~50℃.
․ add 5.4㎖ of37% (v/v) formaldehyde.
․ Mix the agarose solution by swirling and pour the solution into the gel mold.
․ While the gel is solidifying,

2. Sample preparation and electrophoresis
․ Dilute an appropriate amount of 10× MOPS buffer to 1× to be used as running buffer.
․ When the gel is submerged in 1× MOPS running buffer and everything is completely ready, resuspend the dry samples (or RNA in a volume of <2 μl) in 10 μl of formaldehyde gel loading buffer.
․ Heat the sample for 5 minutes at 65°C and then load the gel.
․ Run the gel at 5 V/cm. The ethidium bromide (EtBr) will migrate to the negative electrode and the bromophenol blue (BPB) will travel to the positive electrode.

키워드

생명공학,   노던,   RNA,   DIG