[BIBAC] Electroporation protocols

Thursday, December 04, 2003

Description
A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA. A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation. The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid,and thus replicates as a single-copy plasmid in both E. coli and in A. tumefaciens. The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome. Published examples include a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment inserted into the BIBAC vector and transformed to plants.

Procedure
E.coli
Competent cells
DH10B, prepared as described by BioRad, or Gibco BRL "Electromax" cells
The "electromax" cells are variable (for efficiency and contaminants) and often contain a low level contaminant that grows (slowly) on Kan + Sucrose

BioRad Gene Pulser
Settings:
200 OHMS
capacitance 25 microFD
Voltage depending on cuvettes - see below

Cuvettes
Standard cloning: BioRad (or other) 0.2 cm gap; 2.5 kV;
40 microliters cells

Library construction (highest efficiency):
BioRad 0.1 cm gap; 1.8 kV
22 microliters cells

Protocol
Mix DNA with aliquot of competent cells thawed on ice.
Load cells + DNA into cuvette on ice.
Electroporate, do not put cuvette back on ice. Should get time constant of 4.5 - 4.8 (4.0-4.2 with library construction conditions).
Remove cells from cuvette with microcapillary pipette tip and put into 400 microliters (standard transformation protocol) or 1 ml (for library construction) of SOC.

Incubate at 37 C for 45 minutes (libraries) to about 1 hour (standard cloning).

Selection for Inserts in BIBAC2 (37 C)
Lennox Broth Agar + 40 mg/L kanamycin + 10% sucrose [with electromax cells we use 50 mg/L kanamycin}
Example: 500 mls agar + 50 mls of 50% sucrose autoclaved separately + 440 microliters of kanamycin (40 mg/ml kanamycin

Preparation of Agrobacterium electroporation competent cells

Inoculate 500 ml of Lennox broth (Gibco BRL) with 5 ml of an "overnight" (24-36 hrs) saturated culture in a 2 Liter flask
Shake overnight 250 rpm, at 30 C.
[We usually start the cultures around 3 or 4 PM, and harvest around 9 AM]
Harvest culture at A 600 = 1.5 (1.2 is OK)

Spin down cells at 5000 rpm 10 minutes in sterile 500 ml bottles. Drain well. Wash 4-5X with equal volume of Milli-Q grade water, cold and sterile.

For each wash, add a small volume 5-10 mls of water to the cells and resuspend cells by pipetting gently until no clumps remain. Do this gently with a wide bore sterile pipette. Then add the rest of the water and mix gently.

After the 4th or 5th wash, resuspend cells in 10 mls water and transfer to sterile (we use screw cap) 15 ml centrifuge tubes. Spin at 6000 rpm for 10 minutes. Resuspend cells pellet by adding 1-2 mls 10% glycerol.
You want the total volume of resuspended cells to be 2.5-4 mls depending on the OD that you harvested at.

Aliquot 40 microliters into sterile microfuge tubes on dry ice
Store at -70 C.
Final concentration of cells is 4-6 X 10 to the tenth.

Materials Needed
2L flask with 500 ml LB [We use hand made cheesecloth/cotton tops covered with a piece of foil to seal the flask]
For (1) 500 ml culture: about 3 Liters of sterile cold Milli-Q or other good quality (distilled and deionized) water.
Also sterile centrifuge bottles/tops, and 10% glycerol, cold and sterile.
Dry ice, Sterile microfuge tubes
Electroporation Conditions
We do our electroporations exactly as we do for E. coli, except that the cells recover at 30 C for about one hour. We use a BioRad Gene-Pulser and manufacturer recommended settings and protocol.

We plate out the cells on Lennox broth agar + 50 mg/L kanamycin to select for the BIBAC and 5 mg/L tetracycline for the pCH30 or pCH32 plasmid;

EXCEPT for LBA4404 strains the tet level is 2 mg/L for the helper plasmid.
You should have colonies in 36 - 40 hrs.

Agrobacterium Competent cells

Electroporation protocol same as for E. coli - except recover for 1 hr at 30 C
We typically use the 0.2 cm gap, 2.5V and 40 microliters of cells

Selection for BIBAC2 and derivatives (30 C)
LB + Kan 50 mg/L
Selection for pCH30/32/42
LB + Tet 5mg/L in C58 background, e.g. UIA143, GV3101
LB + Tet 2 mg/L in Ach5 background, e.g. LBA4404

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[BIBAC] Electroporation protocols