3 replies to this topic

[stinky tofu]

Almost every time I purified DNA from overnight bac culture by using Qiagen MaxiPrep kit, the DNA I got in the final step always had some white thing insoluble. I don't think that was DNA. That really troubles me. I am not sure other people ran into the same problem. Any suggestion?

[fred_33]

hi
when i do have an insoluble pellet, it's quite all time RNA. Hence, i treat with RNase proteinase K then phnenol Chloroform.
last year i used to make my minipreps by qiagen columns.
Now that my lab has less money i do not use kits and get better results. My protocol is longer, but i don't mind, and it is cheaper!

Fred

Here is the recipe :
For 50ml culture in apropriate selective media
Spin bacterias 4000rpm 10'
Resuspend in 2ml TpI. Let 5-10' on ice
Add 2ml TpII do not vortex. Let 5' on ice
Add 2ml Tp III

Centrifuge 9000rpm 20' 4°
Discard the pellet and filter the supernatant.

Add 6ml isopropanol. Mix gently. Spin 9000rpm 20' 4°

Resupend pellet in 500µl TE
Rnase / proteinase K treatment : add 1.5µl RNase (10mg/ml). Let 15' 37°
Proteinase K 2.5µl 20mg/ml

Phenol chloroform isoamyl alcohol : add 500µl
spin and discard the organic phase

Precipitate overnight by PEG NaCl : 0.2 ml for 0.5 ml solution
Spin 30' 4° 14000rpm
Wash by EtOH 70%
Resuspend in 50µl ddH20


TpI :glucose 50mM
Tris 25mM pH 8
EDTA 10mM

Tp II : NaOH 0.2 N
SDS 1%

Tp III : 60ml KAc 5M
Acide acétique glacial 11.9ml
Eau 28.5ml

PEG / Nacl : PEG 30% + 1.6M NaCl

[montie420]

The white precipitates you get are from matrix of Qiagen Columns that comes into elute sometimes. Just spin your DNA briefly and collect the supernatant into a new tube.

[stinky tofu]

Thank you guys. That is what I am going to do