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Fosmid Library Construction Service

Frequently Asked Questions: Fosmid Library Construction Service

1. What are the differences between bacterial artificial chromosome (BAC) and fosmid library construction?

2. What are the benefits of using fosmid library construction?

3. What factors influence the quality of fosmid library construction?

4. How to prepare the inserts suitable for cloning into the Fosmid vector?


1.  What are the differences between Bacterial artificial chromosome (BAC) and fosmid library construction?

BAC and fosmid systems are usually used methods for generating large and stable genomic clones for genome sequencing, positional cloning, physical mapping, and analysis of gene structure and function. The BAC genomic library system is used for cloning larger DNA inserts. The insert size of a BAC library commonly ranges from 80 to 200 kb, with maximum sizes up to 340 kb. But for smaller insert sizes of around 40 kb, fosmids are a powerful complementary tool for BACs. A fosmid cloning system employs a low-copy number cosmid vector based on the Escherichia coli F-factor replicon and provides a way for preparing mini-BACs that have an average insert size of 40 kb with high efficiency.

2.  What are the benefits of using fosmid library construction?

1)  Clone & sequencing gaps are nearly eliminated;

2)  Potential problem of generating chimeric clones is eliminated;

3)  Small DNA input is required;

4)  Large fosmid inserts are available (< 90 kb) with a minimal (~10 kb) window of variation;

5)  Exceptional fosmid DNA yield results in a >95% success rate in fosmid end-sequencing.

3.  What factors influence the quality of fosmid library construction?

There are two key steps in fosmid library construction. Firstly, DNA quality is one of the most important factors, which greatly influences the subsequent ligation efficiency that determines the number of fosmid clones. Thus, the extraction of high-quality and high-molecular-weight DNA is critical. Secondly, the size selection of end-repaired DNA is another critical factor determining the insert size of a fosmid library. According to our protocol, the insert DNA recovered is of size ≥ 25 kb. DNA fragments smaller than 25 kb may lead to unwanted chimeric clones.

4.  How to prepare the inserts suitable for cloning into the fosmid vector?

In order to achieve true randomness of inserts, our fosmid libraries are generated by random physical shearing of high molecular weight DNA through proprietary methods. A physically random sheared fosmid library could dramatically reduce the costs for downstream applications, such as screening and end- sequencing.

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