BIBAC LIBRARY Agrobacterium plasmid prep

Thursday, December 04, 2003

Description
Following this protocol, large single copy plasmids (including Ti plasmids) will be isolated as well as any higher copy and/or smaller plasmids. Since in most cases the binary plasmid is higher copy than the Ti, the restriction pattern of the binary will be brighter than the Ti pattern. In the case of a single copy binary plasmid, it may be necessary to do a Southern in order to resolve the pattern of the binary from the Ti.

In any case you will see bands that have come from digestion of the Ti plasmid. Therefore it is essential that you also prep the agrobacterium strain without the binary as a control. If you want to see a nice pattern for the pTi, in my experience NotI works nicely. I have not come up with a method that will CONSISTENTLY isolate small plasmids, but not large ones, so I just use this for everything.

This protocol works very well for C58 strains. Recovery is not as good for Ach5 strains

Procedure
Grow 5 ml overnights LB + drug 36-48 hrs at 30 C
Spin down cultures at about 1500g for 15 min at 4 C (3500 rpm Beckman JA-10 rotor)
Pour off supernatant and keep cultures on ice until all are ready to be resuspended
Resuspend pellets in 200 microliters solution I (50mM glucose, 10mM EDTA, 25mM TrisCl pH 8,5 mg/ml lysozyme added fresh) by vortexing. When all cultures are resuspended, remove each to a 1.5 ml microfuge tube at room temp. Incubate 10 minutes
Add 400 microliters fresh solution II (1%SDS, 0.2N NaOH) invert tubes 4X and incubate 10 minutes at room temp. Invert tubes all at once by placing another microfuge rack on top to hold the tubes in place
Add 60 microliters of fresh alkaline phenol (2 volumes 0.2N NaOH + 1 volume Tris-saturated phenol mixed fresh before using - in my hands this is a single phase) mix by inverting 16X as above
Immediately add 300 microliters of 3M NaAc pH 5.0, mix by inverting 20X
Incubate at -20 C for 20 minutes
Centrifuge 5 minutes at room temp using a microfuge that has slow acceleration and slow deceleration options (such as the "turtle" setting on an IEC MicroMax)
Collect the supernatant and put into a 2.2 ml microfuge tube. If the inversions have been done properly you will be able to remove about 800 microliters. Sometimes there is still a lot of flock and you will only remove about 600. I remove the same amount from each tube.
Add an equal volume of Tris-equilibrated phenol, and extract by inverting 20X
Spin 5 min at room temp "turtle"
Pipette off upper phase into a 2.2 ml microgfuge tube and add two volumes of ice cold (-20) 95% ethanol
Mix by inversion 4X and spin 10 min at room temp "turtle"
Wash pellet with 500 microliters ice cold (-20) 70% ethanol, spin 2 minutes "turtle"
Dry (approximately) 10 minutes in a vacuum desicator
Resuspend (gently) in 40 microliters TE.
Cut 15 microliters for a standard gel, 20 microliters for a pulse-field gel
Gel conditions: 0.7% agarose, 1X TAE buffer 30-40V overnight, or pulsed-field gel

Recipes
LB" = *Lennox L Broth Base (GIBCO BRL) - autoclave
formula per 1 liter demineralized water:

peptone 140 (pancreatic digest of casein) 10 g
yeast extract, autolyzed, low sodium 5 g
sodium chloride 5 g

For agar plates add:
bacteriological agar (2%) 20 g

* for E. coli (growth at 37 C)
* for A. tumefaciens (growth at 30 C)

SOC broth for recovery of cells following electroporation - autoclave
formula per 1 liter demineralized water: bactotryptone 20 g
yeast 5 g
NaCl 0.6 g
KCl 1.86 g
MgCl2 2.04 g
MgSO4 2.46 g
glucose 3.6 g

Following electroporation, the cells are pipetted from the electroporation cuvette into sterile tubes containing SOC media at room temperature

Incubate E. coli @ 37 C for ~ 40 minutes
Incubate A. tumefaciencs @ 30 C for ~ 60 minutes

High sucrose agar medium
For positive selection of inserts in BIBAC vectors (inactivation of sacB gene)

add 100 mls of 50% sucrose (autoclaved or filter sterilized is ok)
to 1 liter of Lennox L Broth based agar
Add required antibiotics taking into account that final volume is 1100 mls.
3-ketoglycoside test for Agrobacterium
Based on a metabolic difference between the Agrobacterium genus and other bacteria such as Escherichia spp and Rhizobium spp. (Bernaerts and DeLey, 1963, Nature 197, 406-7)
Agrobacterium spp grown on lactose make an enzyme called hexopyranoside cytochrome C oxidoreductase that can convert lactose into 3-ketolactose.

3- keto medium
for 1 liter, add to demineralized water: (final)

lactose 10 g 1% lactose
yeast extract 1 g 0.1% yeast extract
bacto-agar 20 g 2%

Streak strain on plates and incubate at 30 C. After colonies appear, flood the plate with a shallow layer of Benedict reagent. Leave at room temperature. A yellow ring of CuO will appear wherever there is 3-ketolactose produced by Agrobacterium.

Note: in our experience the intensity of the yellow produced can vary widely between different Agrobacterium strains.

Benedict Reagent
Sodium Citrate 86.5 g
Sodium Carbonate, anhydrous 50 g

Add distilled water to make 425 mls
With rapid stirring, add 8.7 g Copper Sulfate (CuSO4 o 5H2O) in 50 ml distilled water

Library Freezing Mix (Texas A&M BAC Center)
The colonies are stored in LB with 10% Freezing Mix and the appropriate
antibiotic.

10X Freezing Mix

360 mM K2HPO4
132 mM KH2PO4
17 mM NaCitrate
4 mM MgSO4
68 mM (NH4)2SO4
44% glycerol

We autoclave the Freezing Mix and store at room temperature. It is
normal to see some precipitation after storage. We just mix well before
alliquoting into the LB. We have not had any problems doing this. The
freezing mix-LB-antibiotic mixture should be prepared fresh every few days.

Pipet 50 ul of LB + Freezing Mix + antibiotic per well with a
multi-channel pipettor.

Supplies

Tips

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BIBAC LIBRARY Agrobacterium plasmid prep