The cDNA encoding p6 from the Beet Yellow Virus (BYV) was provided by Peremyslov et al. [27 (link)] and amplified using the primer pair 23/46. This fragment was cloned as an EcoRI/SpeI fragment into pGREEN-CFP, generating the pGREEN p6-CFP construct. The cDNA encoding AtGONST1 (AtGONST1) [54 (link)] was amplified from a total cDNA preparation from A. thaliana Col-0 leaves using the primer pair 24/47. This fragment was cloned as an EcoRI/SpeI fragment into pGREEN-RFP, generating the pGREEN GONST1-RFP. The cDNA fragment Venus-SYP61 was removed from the pUC18Venus-SYP61 plasmid [28 (link)] using NcoI/SalI, and then blunt-ended by Mung Bean Nuclease (Promega, Dübendorf, Switzerland) and cloned between the 35S promoter and terminator of pGREEN-35S, generating pGREEN Venus-SYP61.
Mung bean nuclease
Manufactured by Promega
Sourced in Switzerland
Mung Bean Nuclease is an enzyme derived from mung bean sprouts that exhibits non-specific endonuclease activity. It is capable of cleaving single-stranded and double-stranded DNA and RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using mung bean nuclease
1
Cloning and Characterization of Plant Viral and Organelle Proteins
2
2D CHEF for Resolving Large DNA Fragments
3
DdrC Protects DNA from Nucleases
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The ability of DdrC to protect DNA from digestion by nucleases was assessed on different DNA substrates (supercoiled and linear pBR322, phiX174 single-stranded DNA). Nuclease protection of supercoiled pBR322, linear pBR322 (cut by EcoRV) or ss phiX174 virion was tested with 0.1 U DNase I (Promega), 200 U Exonuclease III (NEB) or 1 U Mung Bean Nuclease (Promega), respectively. 200 ng of ds circular DNA, linear plasmid DNA or ss circular DNA were pre-incubated for 15 min at 4°C in the absence or the presence of DdrC (7 μM, 7 μM, and 2 μM, respectively) in 20 μl of buffer A. 2 μl of the respective 10 X nuclease buffer provided by the manufacturer, and nuclease were then added and the samples were incubated at 30°C for 5 min for DNase I, 30 min for Exonuclease III or 15 min for Mung Bean Nuclease. As a control, 200 ng of ds circular pBR322 DNA was incubated simultaneously with 7 μM DdrC and 0.1 U DNase I. After addition of loading buffer, samples were immediately applied onto 1.2% agarose gels. Electrophoreses were performed in TEP 1X buffer at 4.3 V/cm for 3 h at 4°C.
4
Cloning HOTTIP 3'end into pGL3-Control
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The pGL3-Control plasmid (Promega, Madison, USA) was digested with XbaI (Promega). The long restricted DNA products were recovered and treated with Mung Bean Nuclease (Promega) to degrade single-stranded extensions from the ends of DNA and leave blunt ends. The sequence corresponding to the wild-type HOTTIP 3’end (1561-3840nt) was amplified with HepG2 cDNA using Pyrobest DNA Polymerase (TaKaRa). The PCR primer pair used was: 5’-AAGGCGGTTTTACATACTGGTC-3’/ 5’-TAGCACCTGTAGTTGCCCATTCC-3’. The PCR products with blunt ends were ligated into the appropriately digested pGL3-Control (Promega) containing the firefly luciferase gene as a reporter. The resultant plasmid, designated pGL3-HOTTIP, was sequenced to confirm the orientation and integrity. The HOTTIP reporter gene plasmid with mutant miR-192 binding site or mutant miR-204 binding site was constructed with QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). These mutant plasmids were confirmed by DNA sequencing and named as pGL3-Mut192 or pGL3-Mut204.
5
Validating Hairpin-Forming Oligonucleotide Structures
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Hairpin-forming oligonucleotides (Table 1 ) were purchased from GenScript (Piscataway, NJ), dissolved in water, heated at 95°C for 5 min, and quick cooled on ice for 10 min to facilitate hairpin formation. The Mfold web server with default settings [27 (link)] [http://www.unafold.org ] was used to model possible DNA folding patterns. To validate the predicted secondary structures, reactions containing 10 U of mung bean nuclease (Promega), 500 ng of each DNA, 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, and 100 μg·mL-1 BSA (pH 7.9) was incubated for 1 hour at 37°C then fractionated on a 4% agarose gel.
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