Fast dna end repair kit

The cells were harvested by centrifugation (3000 x g, 10 min) and the genomic DNA was isolated by phenol chloroform extraction or GeneJET Genomic DNA Purification Kit (Thermo Scientific K0721). 60 μg of DNA were fragmented by sonication upon extraction. The following protocol was repeated six times on ice: 2 min sonication with an amplitude of 126 μm/s², cycle setting 5, followed by 2 min chilling on ice (settings according to Bandelin Sonopuls HD2200, sonotrode MS72). Subsequently, the DNA was concentrated using Amicon Ultra 0.5 mL centrifugal filters with a cut-off of 30 kDa. DNA fragments with sizes from 100 to 1000 bp were extracted from an agarose gel and the DNA ends were repaired with the Fast DNA End Repair Kit (Thermo Scientific) according to the manufacturer’s instructions. 700 ng of fragmented DNA were then ligated into the PmeI digested pHORF3 vector (1000 ng) [41 (link)], followed by three transformations per ligation into E. coli TOP10F’ (Invitrogen) by electroporation. The number of required clones to cover the genome was calculated with the following equation [90 (link)]:
$N=\text{ln}(1-P)/\text{ln}(1-\frac{a}{b})$
N = number of required clones
P = probability that a certain fragment is present (set to 99.9%)
a = average size of the DNA inserts (depending on library)
b = total size of the N. gonorrhoeae FA 1090 genome (2,153,922 bp)

PCR-based amplicons 1587 bp in size for the marker PGCRURSE5 were obtained from both parents and purified using a Clean-Up Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. The amplicons were blunted using a Fast DNA End Repair Kit (Thermo Fisher Scientific) and cloned into a blunt pCRScript Amp SK cloning vector (Promega, Madison, Wisconsin, USA). E. coli Top10 chemocompetent cells were used for transformation, and colonies with inserts of interest were picked and sequenced bidirectionally. Sequencing reactions were performed using the BigDye Terminator v3.1 kit (Life Technologies Polska Ltd., Warsaw, Poland), and products were resolved on an ABI3730XL genetic analyser at the Laboratory of DNA Sequencing and Oligonucleotide Synthesis (Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland).

Sequencing libraries were prepared by using a TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) with unique indices according to the manufacturer’s instructions. The 5′ ends of the amplificons were removed by using the Fast DNA End Repair Kit (Thermo Scientific, Waltham, MA, USA), and overhanging adenine residues were added at the 3′ blunt end and then phosphorylated at the 3′ end which prevented the DNA fragment from self-joining. The termination sequence contained library-specific tags (i.e., index sequence) at the 5′ end so that DNA molecules could be modified on the flow cell. PCR was performed on the modified DNA template constructed with the termination sequence to amplify the sequencing library template, and then AMPure XP Beads (BECKMAN Coulter, Indianapolis, IN, USA) were used to purify the enrichment products of the library. The final fragment of the library selection and purification was conducted by gel electrophoresis in 2% gel. The quality of the library assessment was performed on a Qubit 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) system according to the manufacturer’s instructions. In the end, the sequencing of the library was completed and 480 bp paired-end sequences were generated on an Illumina MiSeq-PE250 platform.