Hi, I am trying to prepare peripheral blood for extraction of DNA for cytokines using ficol paque. the method i was given is slightly different to one from amersham, but i have no-one around in labs who's done ever done it!
I have been through first stage fine and got the four layers, then i removed the white cell layer (which is cloudy?) into a fresh tube and added fresh buffer before last spin, but got no pellet at the end. Is there anything obvious wrong or do i need to retry. Am i best to stick directly to the amersham method?
Thank you very much
Katie

Hello,
Your proceedings should be allright.
However, did you pay attention to put your blood very gently on the ficoll layer? The blood must not ... "splash" into the ficoll.
Did you centrifuge at 4°C or at room temperature? I found out that when centrifuging at 4°C you get a better seperation of the diferent phases.
It is indeed the interphase you should recover. It don't know about your 4 phases, I always get 3, the pellet, the interphase with the clouds an the upper yellow phase. I always recover the whole interphase, complete to 50mL with PBS-EDTA and then I centrifuge for 10min at 4°C and 200g.

May be I could help a bit ...

hi,
collect the interphase efficiently, add 30ml wash buffer (PBS or else).
spin at 2600 for 15 min,
discard the wash with a pippette.
check for the cell number.

gud luk

Hi, thanks for your help guys! i'm just about to retry method, i did make sure to not disturb blood and ficol layer, although did it at room temperature (18degrees)! i shall try it at 4 degrees today and lets hope for the best!
Thanks again, I'll keep you posted
Katie

Hi Guys, well i think the reduced temperature helped! definately had more defined phases and at the end had a very tiny pellet! so i removed most of supernatant and transferred to eppendorf and respun and got a small but visible pellet for DNA extraction. is it ok that i did that? it was 5000g for 5 minutes at room temperature. Is there a primer set you can use to check there is definately DNA there, or best just to get my cytokine primers out and see what happens?
Thanks again for all the help
KT

got it working! thanks for the helpful hints!
Katie