Megaprime dna labeling system

Genomic DNA was extracted from rosette leaves using the Wizard^® Genomic DNA Purification Kit (Promega) following manufacturer’s instructions. 750 ng of DNA was digested overnight with 20 units of high fidelity SspI restriction enzyme (New England Biolabs) in Cutsmart^® buffer and electrophoresed through a 1% agarose (w/v) gel for 8 h. The gel was depurinated (10 min in 0.25 N HCl), rinsed, denatured (30 min in 0.4 N NaOH, 1.5 M NaCl), neutralized (30 min in 0.5 M Tris-HCl, 1.5 M NaCl) and capillary blotted onto a Hybond-N + membrane (Amersham) overnight. The membrane was UV-crosslinked at 150 mJ. The DNA probe was amplified from Col-0 DNA with primers indicated in Supplementary Table 2, gel-purified, and labeled with α-³²P-dCTP using the random hexamer priming method (Megaprime DNA labeling system; Amersham) following manufacturer’s instructions and subsequently purified on illustra MicroSpin S-200 HR columns (GE Healthcare Life Sciences). Hybridization was performed using the PerfectHyb™ Plus hybridization buffer (Sigma) following manufacturer’s instructions, with overnight hybridization at 65 °C followed by one washing step (10 min) in 2X SSC 0.1% SDS and two washing steps (15 min each) in 0.5X SSC 0.1% SDS, all at 65 °C. The membrane was imaged on a Typhoon FLA 7000 (GE Healthcare Life Sciences).

Genomic DNA was isolated from cotton leaves using a modified version of the CTAB method [43 (link)]. DNA was purified by ‘DNeasy Plant Maxi Kit’ (Qiagen) for PCR and other molecular analyses.
For the Southern blot analysis, ~10 μg of DNA isolated from each line was digested with appropriate restriction enzymes. λ DNA digested with HindIII was used as a size marker. Digested DNA samples were electrophoresed on a 0.8% agarose gel and blotted on a nylon membrane (Hybond N+; Amersham Pharmacia Biotech). The presence of the different gene cassettes and their integration patterns in the transgenics were studied using appropriate probes. Probes were labeled with α-[³²P]–dCTP using a ‘Megaprime DNA Labeling System’ (Amersham Pharmacia Biotech). Standard procedures were followed for hybridization and washing. Membranes were subjected to autoradiography for 36–48 h at -80°C.
PCR amplification reactions to detect the presence of gene sequences were performed with gene-specific primers (S3 Appendix) using 50 ng of DNA in a reaction volume of 25 μl. The reaction mix comprised 200 μM dNTPs, 12.5 pmol of specific primers, 2.5 U of Taq polymerase, and 1× Taq buffer. The PCR conditions used were as follows: initiation with a 5 min denaturation at 95°C, followed by 30 amplification cycles of 30s at 95°C, 30s at 65°C and 1 min at 72°C. A final extension was performed at 72°C for 5 min.

Two Southern hybridizations were carried out on seven Δsag1 clones and the WT strain that were tested by qPCRs. One μg of each genomic DNA-sample was digested with the restriction enzymes BsaBI or DraIII for 6 h at 60°C or 37°C, respectively. Reaction mixtures were then separated by 0.8% agarose gel electrophoresis containing ethidium bromide. Gels were subjected to depurination (15 min in 0.25 M HCl), denaturation (30 min in 1 M NaCl / 0.5 M NaOH) and neutralization (1 hour in 1 M Tris-HCl, pH 7.5/ 3 M NaCl). Separated DNA fragments were then transferred onto Hybond membrane (Amersham) by capillary transfer and subsequently stably fixed by UV crosslinking for 10 seconds. For blocking non-specific binding sites, membranes were pre-incubated in hybridization buffer (0.5 M Na₂HPO₄, 60 mM H₃PO₄, 7% SDS, 1% BSA, 0.9 mM EDTA) for 2 hours at 65°C.
The DHFR probe was generated from the plasmid P972 by PCR with DHFR forward and reverse primers listed in Table 1, gel-purified and radioactively labelled with α-P32-dCTP using the Amersham Megaprime DNA Labeling System. The labeled probe was heat-denatured at 95°C for 3 min and added directly to the pre-hybridized membranes. After overnight incubation at 65°C the membranes were washed 15 minutes each in 1 x SSC, 0.1% SDS and 0.5 x SSC, 0.1% SDS and eventually exposed to Phosphoimager screens for 20 hours.

Dot-blot analysis for screening disease resistance was performed following Sahu et al. [12 (link)]. Total genomic DNA was denatured by adding an equal volume of 0.6 M sodium hydroxide and an equal amount of each denatured DNA (~ 1 μg) was spotted onto three Hybond N+ membranes (Amersham Bioscience) embedded in a BIO-DOT dot-blot apparatus (Bio-Rad). The samples were spotted in 96-well formats to prepare three identical arrays. The membranes were hybridized with α³²P-dCTP labeled DNA A specific AC1 gene sequence, and then neutralized with neutralization buffer (0.5 M Tris-Cl, pH 7.4, 1.5 M NaCl) for 3 min, washed with 2% standard saline citrate (SSC) and cross-linked using UV cross-linker (Stratagene). Radiolabeled probe was made by random primer labeling method by using Megaprime DNA labeling system (Amersham Biosciences) [12 (link)].

DNA from 21-day-old whole plant tissue was extracted using the Nucleon PhytoPure DNA extraction kit (Cytiva Tokyo, Japan). The extracted DNA was treated overnight with the restriction enzyme EcoRV at 37°C. Next, the DNA was purified by ethanol-precipitation, and electrophoresis was performed on a 1% agarose gel at 20 V for 24 h; the DNA was transferred onto a membrane overnight. Hybridization signals were detected by the Megaprime DNA Labeling System (Cytiva Tokyo, Japan) using probes labeled with radioisotopes.