what could me wrong with my southern blotting - (Oct/26/2005 )

Hi, there,

I just finished a southern blotting and i used ambion's biotinyl kit to label my probe. I performed standard southern blotting and then used ambion's detection system. and then exposed membrane for 1 hour and half before develop. I got nothing!!

it's my first time to do blots. It seems that many things could be wrong but I couldn't figure out what's the most possible.

Plus, the membrane blot was obtained long time ago. Could it be a reason?

please help me out.

thanks a lot

Mac

Is the membrane from Amersham ? some lots of their membrane do not work (alkaline transfer)

the membrane was from Abmion also.
thanks

So this is a "known-good" blot? By that I mean it has been used successfully before, so it's not like it wasn't cross-linked and all your sample washed off or something like that?

How did you generate your probe, and how much DNA did you have and label?

Is there a DNA ladder on the blot (perhaps that was loaded when the gel was run)? You can check your procedure by labeling some of the same ladder and using that as a probe; it should light up the ladder anyway (and perhaps also other things in your experimental lanes, depending on the source of your ladder, the composition of your experimental lanes, the stringency conditions used, etc.).

Hi,

thanks for your reply.

the blot is not a "known-good" blot coz this is the first time I worked with this blot.

my probe is PCR product from plasmid and labelled with Ambion biotinyl kit. First, I tried to use 0.5 nM probe(100ul) to hybridize and got nothing. Thinking that maybe the probe concentration is too low, I used 5 nM to do hybrize again ( I didn't strip the previous probe off the blot), still nothing there. I tested my probe itself and it was well labelled well actually.

so , what can I do?

I would recommend a few rounds of dot-blotting to get the conditions ironed out

perhaps you need more substrate? perhaps you are washing it off somewhere? there can be many places where you are losing sample and dot-blots under variable conditions could tell you the source of your problem without wasting as much time and material

good luck

thanks a lot. But what a dot blotting is?

um...you can probably find a more detailed explanation on a number of websites, but basically you 'dot' a few spots of substrate (whatever you are probing...gDNA or whatever) directly onto a membrane, in different dilutions that you would expect to see bands if your southern worked. Then, you crosslink the membrane and carry out the southern protcol. this saves the running of the gel and the transfer to the membrane.

when you get to the end, you should see a few dots after you expose your film. this tells you that your hybridization and washing steps work right, and that your probe binds well, etc...especially if you add a positive and negative control 'dot' to ensure specificity

if it works, your problem likely lies in the transfer steps and you can trouble shoot from there. if it doesn't work, your problem likely lies in the hybridization, washing, or some other downstream step. this lets you tweak different parts of the protocol in order to obtain signal