Synthetic Genes
Protocol: Handling BACs
BACs (Bacterial Artificial Chromosomes) are synthetic vectors derived
from E. coli F factor plasmids. These plasmids contain a very low copy origin of replication which replicates at 1–2 copies per cell. These vectors
are particularly useful because of their ability to carry large fragmentsof DNA (up to 100s of kb in size) and are less prone to instability andrecombination than high copy number plasmids.

IDT and SGI-DNA Custom Gene Synthesis
IDT and SGI-DNA offer custom gene synthesis, with ordering provided
through the IDT website. MiniGene™ Synthetic Genes can be orderedin lengths from 50–500 bp, and Genes from 501−2 Mb. All Genes andMiniGene Synthetic Genes are constructed using high quality IDT oligonucleotides. Prior to shipping, all Genes and MiniGene Synthetic Genes
are sequence verified. The sequencing method is determined by IDT andSGI-DNA as part of the manufacturing process.
Genes over 5000 bp in length will be provided in BAC vectors, as willsome large genes under that limit. For more information, contact
genes@idtdna.com

Handling BACs
Handling large plasmids requires special care particularly when their sizeis larger than 30 kb. In this size range, avoid vortexing, triturating with asmall bore pipet tip, and other physical manipulations that might resultin shearing of the DNA.

INTEGRATED DNA TECHNOLOGIESTransformation (cont.)
Required Materials
1. Electrocompetent E. Coli, compatible with BACs we suggest:
BAC-Optimized Replicator™ v2.0 & 10G BAC-Optimized Electrocompetent Cells (Lucigen).
2. BAC in ddH2O to a concentration of 0.1–10 ng/μL (See Above:
Resuspending Your BAC)
3. 1 mm electroporation cuvettes
4. SOC media

Procedure
1. Wash and UV sterilize 1 mm electroporation cuvettes, place on iceand prepare cultures tubes with 0.5ml SOC.
2. Thaw cells on wet ice and aliquot 25–30mL to the cuvettes.
3. Add 1 μL BAC plasmid to the tube and mix gently by stirring witha pipette tip.
4. Electroporate using settings of 100 Ohms, 2.4KV, 25mFa.

Purification
The following are important considerations for growing an purifying
BAC-containing E. coli cultures [3].

•You will recieve 1 μg of BAC vector, containing your sequence. Centrifuge the tube for 3−5 sec at a minimum of 3000 x g to pellet the material
to the bottom of the tube.

Due to their low copy number, approximately 10X more culture
must be purified to achieve the same mass of BAC as is typicalwith a high copy vector.

•1. Dilute BAC in ddH2O to a concentration of 0.1–10 ng/μL. It isessential that there is a very low salt concentration in the plasmid
suspension to prevent arcing during transformations.

Small scale preps of BACs can be obtained from as little as 5 mL ofculture using standard alkaline lyses techniques and typically yield
200-400 ng of material [4].

••Larger cultures, of up to 500 mL can yield micrograms of material.

Resuspending Your BAC

2. If the BAC stocks are resuspended in TE buffer, make sure to diluteat least 100X with water, or remove salts with a size exclusion
column or other method.

Growthºº Note that some sequences will be toxic to E. coli upon arabi-BACs contain a chloramphenicol resistance gene and can be selectively
grown in LB medium containing 12.5 μg/mL of chloramphenicol.

nose induction and may result in poor or reduced yields.

ºº Grow cultures to an A600 of 0.2–0.3 in LB in a shaking incubator
at 37°C .

Storing BAC
1. Long term storage of BACs should be in TE pH 7.5–8.0 and at–20°C. Under these conditions, BACs can be stored for periods ofup to several years and still retain viability.
2. For the most stable storage, glycerol stocks should be used [1].

ºº Add L-arabinose to a final concentration of 0.01%ºº Return the culture to the shaking incubator for 3–5 hrs•Several commercial BAC purification kits are available.

ºº Millipore 96-well Plasmid and BAC Preparation Kit.
ºº PureLink® HiPure BAC Buffer Kit.
ºº Nucleobond BAC 100.

Transformation
The best method for transforming BACs is electroporation. The following
protocol a published example for transforming BACs using common lab
reagents and electroporation equipment [2]. Several commercial kits arealso available for subcloning BACs.

BACs can be induced with arabinose to increase their copy number. The efficiency of induction is dependent on the strain of E.
coli used, the size of the insert, and the sequence composition ofthe inserted DNA. In general, small inserts will result in larger copy
number. An increase from 1 copy per cell to 20–40 copies per cellis common with inserts that are smaller than 20 kb.

References
1. Koenig GL. (2003) Viability of and Plasmid Retention in Frozen Recombinant Escherichia coli over Time: a Ten-Year Prospective Study Appl Environ Microbiol. 69(11):6605–6609.
2.

Sheng Y., Mancino V., and Birren B. (1995) Transformation of Escherichia coli with large
DNA molecules by electroporation. Nucleic Acids Res. 23(11): 1990–1996.

3.

Wild J., Hraddecna Z., and Szybalski W. (2002) Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome Research.
12(9):1434-44.

4.

Sambrook J., and Russell D.W., editors. (2001) Molecular Cloning: A Laboratory Manual.
3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.