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Northern blot - How do you do crosslinking - (Jun/22/2005 )

Hi All,

I am not quite sure how crosslinking is done without using a crosslinker. There are three options for crosslinking including baking in a vacuum or conventional oven (30 minutes to 2 hours at 80°C), UV crosslinking or microwaving.

My question are what method is most reliable and how exactly you do it. For example, for baking in a vacuum or conventional oven, how do you place the membrane inside? For UV crosslinking, what distance is between the light source and the membrane.

The reason I ask the questions is recently I had hard time getting positive signals and thought the problem may lie at crosslinking. I had tried baking and UV crosslinking. Protocol book seem not give details how to do crosslinking.

Thank you very much.

-postdoc-

Damn, I have done lots of UV crosslinking for my Northerns... but it was in a Stratagene machine... put your nytran in, push a button and it is done. Never had any problem.

If you don't have that machine, I can't tell you the wavelength, but I can tell you it was 4 bulbs about 20cm (tops) from the membrane and it took about 3 minutes.

I don't like baking my membranes as it makes them very brittle.

-pBluescript-

Vacuum oven - Place membrane between whatman filter paper or any other type of paper
for that matter.

UV crosslink using UV light source - Put membrane on plastic stretch wrap (Saran wrap for eg.)
and place directly on UV for 2-3 minutes.

UV crosslikker (Stratagene) - Again put membrane on top of a pice of paper.

In my experience I find UV or baking in oven is fine. I think the major difference is
that when you have to do several rebrobes, the RNA (in my hands) tend to stay on the membrane
better when the blot is baked. If I am gonna strip and reprobe a membrane 3-4 times I do both crosslinking and UV.

If the signal is weak two suggestions: 1) Always have positive control
2). Maybe try a new probe. Some portions of a cDNA don't always bind with equal affinity or specificity as other regions of the same cDNA.

Best of luck.

-mikew-

Thank you all for the great tips.

Next time I will include positive controls. I use cDNA probe amplified by RT-PCR, purified using qiagen kit and labeled with 32p using random priming kit. Many times it seems I only got signal at 28S and 18S position.

I will keep trying.

-postdoc-

The Stratagene crosslinker exposes membanes to 254 nm UV radiation.

-phage434-

i just put the membranes in a biohazard hood for about 5 - 10 minutes. i guess the distance between the UV globe and the memrbane in about 50cm. i've never had any problems with this protocol and i regularly strip and re-rpobe my membranes without any problem...

-ros-

QUOTE (postdoc @ Jun 22 2005, 11:28 AM)
Hi All,

I am not quite sure how crosslinking is done without using a crosslinker. There are three options for crosslinking including baking in a vacuum or conventional oven (30 minutes to 2 hours at 80°C), UV crosslinking or microwaving.

My question are what method is most reliable and how exactly you do it. For example, for baking in a vacuum or conventional oven, how do you place the membrane inside? For UV crosslinking, what distance is between the light source and the membrane.

The reason I ask the questions is recently I had hard time getting positive signals and thought the problem may lie at crosslinking. I had tried baking and UV crosslinking. Protocol book seem not give details how to do crosslinking.

Thank you very much.


One more option.....

I used to crosslink using the autoclave. I remember having to turn the temp down (can't remember to what) and then doing a 10 min dry cycle to crosslink. Seemed to work fine. Sorry I can't be more help on the temp, but someone down the hall got the Stratagene crosslinker and, well you know the rest...

Cheers,

LTR

-L_Reiter-