Decaprime 2 dna labeling kit

Total cellular DNA was prepared according to Løbner-Olesen and von Freiesleben [60 (link)]. DNA was digested with PvuI, and fragments were separated on a 0.7% agarose gel, transferred by capillary transfer to a Hybond-N⁺ membrane (Amersham Pharmacia Biotech), and probed with an approx. 1 kb NKBOR fragment, which hybridize to NKBOR. The probe was prepared by PCR amplification using primers NKBOR_Probe_FW and NKBOR_Probe_RV (S7 Table) using pNKBOR as template and labeled with [α-³²P]dATP (Amersham Pharmacia) using the Random Primer system (DECAprime II DNA Labeling Kit; Life Technologies).

Gene-specific probes used for RNA blot analyses were designed as SIZ1-1, SIZ1-2 and Actin 8 that have lengths of 1056 bp, 663 bp, and 502 bp, respectively. The pHL080SIZ1 construct was used as the template to amplify the two OsSIZ1 fragments (i.e. SIZ-1 and SIZ-2) via PCR. An Arabidopsis cDNA library was used to amplify the Actin 8 gene. Probe for DNA blot analysis was made by using the OsSIZ1 gene fragments amplified by PCR. Two PCR products of OsSIZ1 were gel-purified and mixed in equal amounts and then used as templates for making probes. Random priming reaction was performed with the DECAprime™ II DNA Labeling Kit (Life Technologies) to radio-chemically label the PCR fragments using ³²P-α-dATP as the ³²P donor. The oligonucleotide primers used were ACT8_F and ACT8_R for Actin8, SIZ1-1_F, SIZ1-1_R and SIZ1-2_F, SIZ1-2_R for OsSIZ1. The hybridization conditions for both RNA blot analysis and DNA blot analysis were essentially the same as those described by [45 ].

For the deletion of the trpA gene in HV31 (44 ), the strain was transformed with plasmid pTA131-Cupdo (34 (link)), which contains the up- and downstream regions of CRISPR-locus C for integration into the genome. Subsequent selection for loss of the pyrE2 marker by plating on 5-FOA agar led to pop-out colonies. To confirm the removal of trpA, Southern blot analyses were performed. Genomic DNA from potential deletion mutants was digested with SacII, separated on a 0.8% agarose gel, and transferred to a nylon membrane (Hybond^TM-N, GE Healthcare). A 250-bp fragment of the downstream region of locus C was amplified using primers Cdeldoi and DomitteC, radioactively labeled with [α-³²P]dCTP and the DECAprime II DNA labeling kit (Life Technologies) and used as hybridization probe. The resulting strain was termed HV32.

Total RNA was isolated if not stated otherwise from exponentially growing H. volcanii cells as described (31 (link)). After separation of 10 μg of RNA (total RNA) on a 0.8% denaturing agarose gel or 8% PAGE, RNA molecules were transferred to nylon membranes (Hybond-N⁺ or Hybond-XL, GE Healthcare) and incubated with radioactively labeled DNA fragments. PCR fragments were labeled using a DECAprime II DNA labeling kit (Life Technologies). Oligonucleotides used as hybridization probes were radioactively labeled at the 5′ end with [γ-³²P]ATP. To quantify the amount of RNA the membranes were exposed to imaging plates (BAS-MS, Fujifilm) and analyzed using the FLA-3000 scanner (software BASreader 3.14). The intensity of signals was measured with ImageJ. The signals of the bgaHa, β-lactamase transcripts, and RNase P RNA were put into relationship to the 16S rRNA signal, which was used as RNA loading control. To obtain the percentage of RNA in the strains expressing crRNAs, the amount of RNA (mRNA and RNase P RNA) in a strain expressing a non-targeting crRNA was set to 100% and the RNA amounts in the strains containing the crRNA were set in relationship to these data. All Northern blot analyses were performed at least three times.

To delete the bgaH gene from HV32, the strain was transformed with the plasmid pTA617 (41 (link)) (which contains the up- and downstream regions of the bgaH gene) to achieve integration of the plasmid into the genome. Subsequent plating on 5-FOA agar generated pop-out colonies that were analyzed by Southern blot analysis. 10 μg of SalI-digested genomic DNA were separated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond^TM-N, GE Healthcare). Using primers bgaHKODO-for and bgaHKODO-rev, a 400-bp fragment of the downstream region of bgaH was amplified. The fragment was radioactively labeled with [α-³²P]dCTP and the DECAprime II DNA labeling kit (Life Technologies) and used as a hybridization probe. The resulting strain was termed HV33.

For deletion of the cas6b gene, HV29 was transformed with the plasmid pTA131-cas6updo (32 (link)) to achieve integration of the plasmid into the genome. Subsequent plating on 5-FOA generated pop-out clones that were further investigated by Southern blot analysis. 10 μg of SaII-digested genomic DNA was separated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond^TM-N, GE Healthcare). A 300-bp fragment of the upstream region of the cas6 gene was amplified using primers Cas6Sonde-up and iPCRCas6KOup2. The fragment was radioactively labeled using [α-³²P]dCTP and the DECAprime II DNA labeling kit (Life Technologies) and subsequently used as hybridization probe. The resulting strain was termed HV30.