Pierce North2South Direct HRP Lebelling and detection system

[Donya]

Has anyone used this kit? It has been discontinued, and I was wondering that if you have used it in the past, and still require to carry out the same sort of hybridisation what do you use instead? Pierce has an incredibly expensive kit that it markets instead (Cat#: 17175), and from what I can tell, requires a lot more work.

[Donya]

sorry that should be Labelling not Lebelling!

[admin]

Hello Donya,

that kit is a bit expensive as it uses commonly available reagents.

Roche Biotin labelled nucleotides are about 200$ but you can make a ton of label. I used T7 RIBOMAX from Promega to make a few mgs of RNA for about another $200.

The problem is to detect it well you have to use the Lightshift Chemiluminescent kit from Pierce which kind of expensive....

You can still make your own experiment using separate suppliers products as I did.



How much is the Pierce North2South kit?

[Donya]

Hi admin,

The North2South Pierce kit that both labels and detects (Cat#17175), which is really a combination of two other kits, is $999 (Australian).

If I were to buy the two kits from them separately, it would be Cat#17095: $754 and Cat#17075: $564. So its cheaper buying it all together. But a grand for a labelling and detection system just sounds rather expensive to me. Its a real pity that they discontinued their Direct HRP Labeling and detection system a few years back, i think that would have really been what i was looking for.

So I started to look around, so far ive found the Roche Applied Science
DIG Labeling and Detection Starter Kit. I havent really properly read up about it yet and at this present moment cant recall the difference between kit I and kit II. Its probably because i really havent read the manuals on either..yet.

The Kit I (Cat#7145832910) goes for $559.
and Kit II (Cat#11585614910) goes for $567.

I wont be needing to make mg of RNA, but I will need to make ug of mRNA, which I will be using in the Clontech cDNA substraction kit, which going by the publications seems to be the way to go if youre performing a cDNA subtraction hybridisation. The thing is that this method can give a high level of false positives, so I need to use a dot blot type of assay to weed out the false positives. This is where the Pierce detection systems come in, to allow labelling and detecting a set of different probes, to verify positives as true ones.

[Donya]

Sorry I gave the wrong Cat# for Kit I, its 11 745 832 910.

The difference between the two kits is that I is colourmetric, whereas II is chemiluminescence based. I was leaning towards the colourmetric Kit, since then I wont have to buy X-ray film and all the solutions that go with developing it. It appears however, that the chemiluminescence kit is more sensitive, but I dont know whether this is worth the trouble in my case!

Sensitivity difference:

Kit I: A single copy human gene is detected in a Southern blot of 1 ug digested placenta
DNA.
Kit II: A single copy human gene is detected in a Southern blot of 0.3 ug Bgl II or Eco RI digested placenta DNA.