Sorry i don't understand what is exactly the problem and what you want to do.

:O)

This phenomenon seems to be quite common with different ladders. At least for those from New England Biolabs they say the ladder bands contain pUC-like sequences in them, so probes generated from a pUC or PBR-based plasmid could have some level of plasmid contamination in the probe that hybridizes to the ladder bands. So in your case, probably using a PCR-generated probe would reduce that source of contamination.

Now, I've heard of this more as a problem, since DNA in the ladder is usually in great molar excess compared to that in the samples, and sometimes very strong signal from the ladder can mask that from samples (or just make your southern look not-so-nice). This is the first time I know someone who actually wants those bands to appear. In any case, you can always mark in pencil the ladder bands after the transfer for reference, so it doesn't really matter if they hybridize or not...

Hi Karina

You can resolve the problem in few ways given below

1) As suggested by Gustavo above you can make marks on the blot with the pencil for reference

2) You can also take a gel phorograph before the transfer on a set zoom and distance..and later take photograph of the autoradiogram on the same zoom and distance ...and superimpose them

3) If you rally want to use and also detect the DNA ladder on autoradiogram.....I will suggest you to also label your ladder with random primer labelling......cut the blot infor half after the transfer.....hybridize one containing samples with the probe and the other one containing the marker lanes with the marker probe....and develop them

but this will make your work double.....if it really needed......the first two options will give you the information needed....

bye

There is also a huge molar excess of ladder (several tens of nanograms for each band). In contrary a 10-kbp fragment represents only 1/300 000 of the total genomic DNA (3xE9 bp). Imagine that you use 10 micrograms of genomic DNA for a Southern blot (this is the amount of genomic DNA from approximately 1 000 000 cells). In these 10 micrograms of genomic DNA there are only 33 picograms of such a 10-kbp fragment. Thus each band from the ladder is in a very high molar excess and can be non specifically revealed due to a "mass effect".

Hi,

thanks for your answers!

Gustavo, I sometimes mark the ladder with a pencil when I do a Western Blot, but actually I never used DNA ladders in an agarose gel that are prestained and thus visible on the membrane after the blot. If you know some, could you tell me where to get them?

And for me it was always very nice to see the ladder in my southern, because I exactly new which size my labelled bands were. Of course I used only a little amount of DNA ladder, so that the signal did not mask my actual signal.

My actual basic question was if it is really true that the ladder will not be labelled when I use a PCR derived probe. Sorry for my confusing description!

The idea I had is putting a ruler next to my gel when I take the picture of the gel (so before blotting), so that I now the height of my ladder in cm.

I will let you know how it worked and if my ladder was labelled with my PCR derived probe!

Cheers,

Karina

Hi Karina,

If you are using enough amount of ladder you should be able to see the bands when checking your membrane on the UV after the transfer, and then mark them with pencil. Using the ruler for the picture is a good option, and if you are marking also the wells with pencil, that should be enough reference for the position of your sample bands. Ajay's suggestion of taking comparable pictures of gel and autoradiogram sounds also great.

Good luck!

>My actual basic question was if it is really true that the ladder will not be labelled >when I use a PCR derived probe.

As Gustavo said, if you were seeing your ladder on the autoradiogram before, perhaps you were also labelling plasmid contamination as well as your probe, and so happened to also hybridize plasmid sequence with your ladder. Were you separating your probe from the left-over plasmid before?

If you are now making probes by PCR any template DNA can (likely) be ignored since you have a vast excess of the PCR product. So unless you happen to be amplifying from a plasmid and using primers that also amplify plasmid sequence, you should not detect the ladder any more.

Unless I've misunderstood and you are actually adding the radionucleotide to a sample of your ladder and labelling the ladder. I'm assuming you meant something in your probe was hybridizing with your ladder sequence when you were using probes from a plasmid.

I always making probes for Southerns by cloning a PCR product into a vector using a PCR-cloning kit, making mini-preps and and isolating the fragment from the vector . In this way you can see ladders, as a very small amount of contaminated vectors hybridize to the ladders. If you use a PCR product directly as a probe, you won't see it.

Please find some of my publications for Southern blots.

http://www.nature.com/ng/journal/v37/n3/full/ng1515.html

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0038958

http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1000472

HI, in Southern blot ladder is not needed in gel pic. you can ark them later. If the ladder is labeled with probe no problem. you can erase that using Adobe Photoshop.

Hey guys,

it worked out making a photo of the agarose gel with a ruler, but from my point of view it is so much easier to use a probe that was cut out of a vector and then see the ladder in the final picture... I can only recommend this technique, for me it was always more helpful than annoying (so far...).

Cheers,

Karina

Hi Karina Oberheide,

I have done a lot Southern blots using PCR products to make probes and I could see the clear ladder in the final picture (attached file). You can try GeneRuler1 kb DNA Ladder. It worked well for all my experiments.

Best,

Phat