southern blot - (Sep/16/2006 )

hi everybody

we are starting new in our molecular lab ,,, we were thinking of establishing southern blot ( which we hadnt dealt with before ( i know this is primitive for most of u ( if not all )
i was woundering ... can i do it alone if i hadnt practiced it before ? and what are the requirments ??? and what well be the aproximate cost???

where are you starting from?

radioisotope alpha-32PdCTP from amersham cost 160 GBP. Due to the high cost and short working life of said isotope 4 labs along with mine share the same bottle of isotope and take turns buying in more stock. A roll of Hybond-N+ (20 cm × 3 m) (nitrocellulose sheet) cost another £197 (amersham)

And there is the hybridisation incubator... I have no idea how much that cost. You need an extra set of pipette for hot work, a hot centrifuge and hot waterbath for probe building abd a prespek shield with table top covering. Communal stuff would be a geigercounter, a hot lab/room and Waste disposal for hot waste.

But aside from these things, the rest of the reagents (buffers, denaturation buffers) are cheap if you make them yourself. (My lab's transfer tank is self made)

As for doing it yourself, well it is a little of an art to guess how much labelled probe to use. The hyb temperature is fairly robust.. unless you are doing something difficult... such as diffentiating between similar sequence. But if you can get yourself a very detailed protocol, you should be okay.

thank u for the detailed explanation ...but it made me more depressed ...it seems so complicated

i have experience in thermocycler PCR ...RT PCR ,,,,butnot with REAL TIME pcr OR sOUTHERN BLOT

i dont know seems so complicated but its crucial for ourlab to florish

Don't worry, Southern blot isn't complicated.. It is just very equipment intensive to set up. And takes a long time to do. 2 days to do the wet work and maybe 4 days (at -80 Celsius) for exposure.

P.S, I forgot the Xray film, Xray cassette, and Xray developer.
Though my lab develops our films by hand with trays of the three solutions.. talk about nerve racking with the developing solution. (Which needs a darkroom)

And yes, all can be replaced by the photoflorencense imager. But I no idea how much that thing cost.

thanks alot perneseblue for your cooperation

and for telling me about the films too i wounder how much that costs

we are building a low dudget lab ( from the scratches ) hope we can pull it through

I am not sure about the price...but I do know the films my lab uses comes from Kodak. As well as the developing solutions.

lula, I have a southern method that takes about two to three days. it involves a few kits in part, it's scrapped together with some Maniatis stuff, and from my own experience. it was the cheapest way to set it up, for our lab. would you like me to PM you a protocol?

by the way, it's non-radioactive

hi aimkines

long time no see ...

ofcourse i would like u to send it to me i apprecaite the kind offerr ....thanks

what do u need ??? my email ???

I will send it by PM later today

I need to type it in and will do it between assays

thanks alot ...you are really kind
thanks for your cooperation