Scaling BAC on Time and Sample

A new and simplified quality control method confirms the cloning of both small and large inserts in bacterial artificial chromosomes (BACs) with significantly less time and sample.

The Scientist Creative Services Team in collaboration with Agilent Technologies
Apr 9, 2021

Bacterial artificial chromosome (BAC) systems offer numerous benefits. These roomy vectors can contain an entire gene and all of its associated regulatory elements, allowing scientists to easily manipulate gene expression. In addition to housing large genes, BACs facilitate de novo sequencing projects in a number of species, from microbes to humans. However, before you can use BAC systems to fulfill your DNA sequencing needs, you must first run the necessary quality control experiments. These essential assays guarantee that your gene of interest and all of its machinery successfully inserted into the BAC clones. 

For studies using BAC systems, researchers often use pulsed-field gel electrophoresis (PFGE) to separate DNA fragments. Unfortunately, PFGE takes a long time to run. Clocking in at around 16 hours, it is far from a quick quality check. When used alongside the Agilent Technologies pulsed-field capillary electrophoresis Femto Pulse system, the new 165 kb BAC Analysis kit takes less than 3 hours. In fact, a run of 12 samples can be analyzed in only 170 minutes. 

Does saving time sacrifice the quality of the fragmentation process? A major limitation of classic PFGE systems is poor fragment resolution, especially for larger fragments. The 165kb BAC Analysis kit is designed for samples containing DNA fragments spanning a range of sizes from 75 bp to 165,000 bp. To examine the precision and accuracy of DNA fragmentation, Agilent scientists recently compared DNA samples separated by Femto Pulse using the 165 kb BAC Analysis kit to those separated by a classic PFGE system (Pippin Pulse).

BACs run with the 165 kb BAC Analysis Kit and the Femto Pulse system exhibited all of the expected peaks within the sample and clearly separated all 23 DNA  ladder fragments. However, the traditional PFGE method struggled with fragment resolution as only half of the ladder fragments appeared on the gel. While it was unable to separate the larger 24 and 24.5 kilobase fragments in the DNA ladder, the smaller fragments between 75 and 1,500 bp did not even appear on the gel. The same poor separation was observed within samples. Smaller DNA fragments appeared to be larger as they were unable to completely resolve, and a 20,000 bp fragment was almost impossible to detect. The inability to detect smaller DNA fragments is likely due to their lower DNA concentrations. The 165 kb BAC Analysis kit run with the Femto Pulse system provides superior resolution and sensitivity capable of separating and detecting even minimal amounts of DNA in a fraction of the time.

Not sacrificing the quality of your experiment to get quicker run times is definitely a bonus, but maybe you have other concerns about your PFGE system. After spending so much time preparing your BAC library and dozens of clones, the last thing you want to do is use up all of your sample on quality control studies. What you need is unparalleled detection sensitivity. The new Agilent 165 kb BAC Analysis kit quickly delivers DNA fragment resolution with only picograms of a sample. According to a recently published technical note, the 165 kb BAC kit needs only 1.6 to 50 pg/μL for single fragments. 

So, whether you are looking to use less of your valuable sample, improve the accuracy of DNA separation, or maybe just save some time, the new Agilent 165 kb BAC Analysis kit along with the Femto Pulse system is designed to achieve all of these goals.

For Research Use Only. Not for use in diagnostic procedures.