concerns of the week_11 May

There haven't been many questions lately so I am going to post some of the concerns that were discussed in classes when we revised for our topics through our Biology Discussion Forum, which visitors from other classes might be curious about too.

Does the loading dye stain the DNA/proteins?

No. The loading dye does not stain the DNA/protein but serves to weigh down the DNA/protein and also for the tracking of electrophoresis progress.

The loading dye is typically lighter than the fragments you want (you can purchase loading dye according to molecular wts)so by observing the progress of the loading dye front (colored), we will know when to stop the current (that is running through the buffer) - typically when the front reached the end of the gel.

Will charges in the ionic buffers affect the movement of the fragments? Will DNA be neutralised by the positive ions (asked by FLEA XW)
- I forgot to get back to the class on this

After a deep ponderous moment, I shall say no. Like electrochemistry, the positive ions will be drawn to the cathode while the negative ions will be moving towards the anode. so the positive ions do not neutralise the DNA but will be heading towards the cathode which bears a greater attaction (greater charge). Similarly, the repulsion between negative charges is neglible as all negative ions/fragments are drawn to the anode.

PS: I am taking back my words on repulsion btw SDS and DNA. It is definitely more redundant should it ever be done.

Probes *ouch*

Probe are technically useless unless you label them.
you can use a radioactive probe or fluorescent probe, both of which are typical.

Southern Blotting

Apologies to KR. In Southern Blot you do have to use nitrocellulose membrane. After running the gel, the fragments will be transferred to a nitrocellulose membrane before probing.

- Is DNA probe used all the time? What if we are doing a protein separation?

I think some of you are familiar with Western Blot (for proteins) but most are not.
Pls do not make the mistak: if you are running Southern blot, you are working with DNA fragments, therefore you can use a DNA probe to determine the presence of a specific DNA seqences in any of the ethidium bromide stained bands.

If you read your notes carefully, we stopped at use coomassie blue to stain for (all) protein bands. If you want to verify the identity of any bands (if it is made up of a particular protein) we would use antibodies that will bind specifically to the protein of interest. Again, the protein fragments on the gel have to be transferred to a nitrocellulose membrane before probing with the antibodies can be done.

http://en.wikipedia.org/wiki/Western_blot

Cheers! I think the rest are okay yah? =)