northerns...... - (Nov/03/2001 )
Hi all, I am a graduate student who has just started doing northerns. I am encountering the following problems:1. I see novery faint signals on my blot,while my dot blots light up beautifully. The signals on the northern transfers are seen only after three days versus one day for the dot blot,that too for the highest conc of RNA.2. there is at least a 100 fold difference between similar amounts of RNA in the transfer versus dot blot.I have done the following: a. All the reagents and glass ware are clean- baking, DEPC, handling with gloved hands etc.b.Ensured the integrity of RNA- can see both the 18s and the 28s bands clearly.c. Run a formaldehyde gel,transfered by capilliary transfer overnight onto zetaprobe (biorad- a charged nylon membrane)with 10XSSC. Following transfer, the gel on etbr staining is absolutely blank. What do you think is causing the faint bands? I have also confirmed the transfer by running the probe fragment (cold) in the same gel- it lights up very well.Thank you all for going through this lengthy mail and your responses in advance.Lakshmi
You can increase the concentration of RNA in the gel. Generally 15-20 ug of RNA is good enough to see rare mesages, however, relatively abundant RNA can be seen even with lower amount of RNA. So you need to adjust your concentration of RNA depending upon the amount of your target RNA in the cells. If the RNA quality is good as you see on the gel, the northerns should come out OK. How about a housekeeping RNA like GAPDH or b-actin. Do you get faint band for that too? if not, then your taget mesage may be low and by increasing RNA conc., you should be able to get better bands.Hope it helps.