Agrobacterium plasmid DNA miniprep

Monday, November 10, 2003

Description
Agrobacterium plasmid DNA miniprep

Procedure
1. Inoculate a single colony into 2 ml of LB or YMB containing appropriate antibiotics.
*Set up 3 or 4 cultures and combine the cells.
2. Incubate shaking at 28oC until culture reach stationary stage.
3. Spin 1.5 ml of cells in 4 or 5 Eppendorf tubes for 3 min in a microfuge at 14,000rpm.
4. Remove the supernatant and resuspend the pelletoin total volume of 100 ul of ice cold Lysis buffer, voretex.
*Add small spatula Lysozime to 1 ml of Lysis buffer immediately prior to use.
Break up the pellet by drowing the solution up into a Gilson repeatedly.
5. Vortex for 10 sec and incubate at RT for 30 min.
6. Vortex, add 200 ul of 0.2M NaOH, 1.0% SDS. Mix by gentle shaking. Make 0.2M NaOH, 1.0% SDS immediately prior to use.
7. Incubate at RT for 10-30 min (the longer the incubation the better) Add 150 ul of cold 3M NaOAc, pH 4.8. Mix by shaking and incubate on ice for 5 min. Should go white and fluffy.
8. Spin for 5 min in a microfuge at 14,000rpm. Should see white scum on surface.
10. Remove supernatant to a fresh tube and add an equal volume of phenol : chloroform 1:1. Mix by shaking.
11. Spin for 5 min in a microfuge at 14,000rpm.
12. Remove top phase to a fresh tube. Add 1ml of absolute EtOH -20oC
13. Remove the EtOH and wash the pellet in 1 ml of 70% EtOH -20oC
14. Spin for 3 min in a microfuge at 14,000rpm.
15. Remove EtOH and dry pellet under vacuum.
16. Resuspend the pellet in 50ml TE buffer. Mix in pellet by drawing up onto a Gilson repeatedly.
17. Incubate at RT for 5-10 min. Add 1 ml of 1 mg/ml-1 RNAse.

Recipes
LYSIS BUFFER
50mM Glucose
25mM Tris-Cl pH 5.8
10mM EDTA
4mg/ml-l Lysozyme

Supplies

Tips

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Agrobacterium plasmid DNA miniprep