Plasmid DNA Isolation problems, contaminating chromosoma DNA - How to isolate large inserts by miniprep and without chromosomal smearing (Nov/23/2006 )

Hello
I am having problem with isolating my cosmid by miniprep method of Sambrook/maniatis. What I get is very low yield (very small band while i cannot use the rest for restriction digestion) with some chromosomal smearing. I have used different volumes from 3 ml to 10 ml to isolate it but having similar problems.
Is there any method to isolate large insert (mine a cosmid clone so it is more than 40 kb in size upto 52 kb). Can i do minipreps in Microtiter plate for such clones.
I will appreciate any help.
Thanks in advance.

There are special kits for cosmid and BAC isolation which you might want to look at. Also consider chloramphenicol plasmid amplification, which will increase the yield of plasmid from cells. You could also consider the pSCANS series of plasmids from BNL, which are normally low copy number and inducible to high copy number for plasmid preparation.
Your miniprep method may not be good for cosmid length plasmids. Saying "Sambrook/Maniatis" is like saying "molecular biology" and gives very little information about what you are really doing.

i use that method for extracting bacmids.

i've used this method for bacmid preparation (roughly 135kpb DNA)

2ml culture with appropriate selection
do not vortex, pipett always gently, do not freeze thaw (but 2cylces don't affect prep quality)
put in eppi. Spin 1' 13000g
resuspend cells with 0,3 ml solution 50mM Tris HCL pH :8,0 ; 10mM EDTA ; 100 µg/ml RNase (P1 prep quiagen)
add 0,3 ml d’une solution 0,2 N NaOH, 1%SDS, homogenize gently.
Let incubate 5-10' RT
add 0,3 ml of solution Na acétate 3M homogenize gently during addition.5-10' on ice
spin 10 min 13 000g R.T.
Put 0.8ml IprOH and add the supernatant of the spinhomogenize slow by inverting tube.
5-10' on ice and spin 15' RT 13000g
wash by 0.5ml ethanol 70% twice
13 – Centrifuger 5 min à 13 000g
Let dry 5 10 min R.T
Dissolve in 40 µl TE.
CONSERVER LES BACMIDES A 4°C (ne pas congeler)

you also may have a look here :

isolating large plasmids, is it possible