RAPD analysis with not that pure DNA - (Oct/25/2010 )

I am a master student doing my master project on RAPD with Algae.
the problem is i always couldn't get pure DNA to further my RAPD analysis, now i am still in the optimizing section for my primer(OPERON),
and my supervisor is not that happy with my slow progress......

since i got a A260/ A280 ratio lower than 1.8, so i decided to do phenol:chloroform extraction.
but unfortunately i got stuck here.....

i prepare my phenol chloroform solution by melting phenol crystal and then buffer saturate it.
then i follow the general P:C extraction protocol till the end, but......

the problem i face during the extraction is, i cannot see the aqueous phase, all i can see is just the phenol underneath, and another milky layer on top, although i read through most protocol it should have three distinctive layers.not even the slightest thin layer, nope, na-da .....


i cannot understand how i cant get that third layer.....
can anyone shed some light on me ? thanks alot

As far as I remember DNA quality can be a major issue in RAPDs, i.e. changing quality/purity might change amplification and therefore band patterns...
Anyway buy the phenol chloroform mixture premixed from a company, they're also buffer saturated and ready to use, Sigma Aldrich and other companies offer them.... preferable to reduce the risks of phenol (dust) and avoid problems with the preparation of the mix.

I hope you have sorted your extraction problems out. I always used to add a small amount of silicone to my extraction tubes which then forms a more solid layer at the interface so you can easily remove the aqueous layer. When I was doing RAPD I found that the constituents of the PCR buffer had a great effect on the profiles produced. I used the buffer matrix in this paper

Paper

and then chose the buffer that gave the best and most reproducible results. I would really recommend optimising your PCR buffer.

thanks ....
i will update bout my progress .....
hope everything will turn out fine .....

this is what happen during my phenol chlorofrom purification,

when i took out my samples from the centrifuge,
i found out that i have to let the sample to settle for few minutes before i can really sees a milky interface...
but is this normal, coz my protocol did not mention bout the settle down time needed.

-here is my protocol


1. add equal amount of p:c to dna solution(i use water).
2. mix weel and centrifuge for 10000rpm for 5 mins.
3. pipette out aqueous layer to new tube .
4. add equal volume of chloroform
5. spin n pipette out aqueous layer(here also i found that there will be another milky layer if i let it settle down for few mins after taken out from the centrifuge)
6. then do ethanol precipitation to precipitate the dna.

but after i did the purification, i still get low a260/280 readings, and the dna amount is even lower.....
i dunno where i did wrong.....would appreciate much if anyone can show me my fault.....