An overview of the procedure is provided in figure 1. Can someone provide a protocol for dnase i treatment after. Dnase i deoxyribonuclease i digests single and doublestranded dna to oligodeoxyribonucleotides containing a 5 phosphate. Deoxyribonuclease i, filtered filtered through a 0. Preparation of dnafree rna prior to rtpcr and rtqpcr 2, see protocol on reverse page. Pretreat rna with dnase i, amplification grade cat. To ligate adaptors i and ii, digest with mme i, and to construct dnaseseq library follow steps 2843 described in protocol for mammalian cells in song and crawford 2010 6. Invitrogen dnase i, amplification grade fisher scientific. We get good results with the cdna synthesis after weve used this dnase kit. Dnase treatment following thawing of cryopreserved pbmc is. Ultrapure dnasernasefree distilled water from invitrogen. Dnase i, amplification grade, is suitable for eliminating dna during critical rna purification procedures such. Dnase i, rnase free is an endonuclease that nonspecifically cleaves dna to release di, tri and oligonucleotide products with 5 phosphorylated and 3 hydroxylated ends. Dnase i, recombinant, grade i, originally isolated from bovine pancreas, is a recombinant enzyme expressed in pichia pastoris.
Transfer thawed cells into a sterile 50 ml conical tube. Having dealt with heavily contaminated samples, i used this higher amount of dnase and got complete removal of dna from my rna of interest. Protocol for dnase i treatment of rna molecular biology. Determination of rna concentration and rna integrity number rin the rna concentration in individual rna samples was determined using the rna pico 6000 labchip kit agilent technologies. The final dnase iproteinase k protocol consisted of the following steps. Rna concentration and integrity were evaluated using an agilent 2100 bioanalyzer and an rna 6000 pico assay kit. We offer a wide variety of pcr enzymes, master mixes, and pcr protocols to meet your experimental needs for routine pcr, qpcr, or rtpcr. Dnase i synonyms, dnase i pronunciation, dnase i translation, english dictionary definition of dnase i. Dnase i acts on single and doublestranded dna, chromatin and rna. Dnase i is a dnaspecific endonuclease that hydrolyzes ds or ssdna. The following protocol for thawing frozen samples outlines how to prepare a single cell suspension and minimize cell clumping using dnase i.
Prepare duplicate tubes if positive and negative reverse transcriptase rna samples are to be used in the amplification reaction. The dnase treated rna can then be used immediately for the reverse transcription reaction. Invitrogen ambion dnase i rnasefree fisher scientific. Design primers that anneal to sequence in exons on both sides of an intron or at the exonexon boundary of the mrna to differentiate between. The same protocol can be used for rna extraction from cell cultures. Dnase i, recombinant, grade i from bovine pancreas. This product is not intended for the diagnosis, prevention, or treatment of a disease. Rna isolation with trizol invitrogen and qiagen rnaeasy. I tretated my rna preparation with dnase 1 according to the protocol ut the rna got degraded. A typical dnase i reaction protocol m0303 protocols. Turbo dnase cleaves doublestranded dna nonspecifically to leave 5 phosphorylated oligodeoxynucleotides. Turbo dnafree kit turbo dnase treatment and removal.
That one has an edta deactivation step, using 1ul of 25mm edta for each 10ul sample. Can someone provide a protocol for dnase i treatment after rna. Dna labeling by nicktranslation in conjunction with dna polymerase i 1, see protocol on reverse page. Monarch rnase a degrades singlestranded rna at c and u residues. Deoxyribonuclease i worthington biochemical product catalog. Invitrogen ambion dnase i rnasefree nonspecific endonuclease that degrades double and singlestranded dna and chromatin.
Lysed samples were bound to a silicabased filter, treated with deoxyribonuclease i dnase i, and washed several times before elution. Print bookmark share for dnase treatment with qiagen or preanalytix rna purification kits pdf 35kb english format. Add the dnase buffer as well as the rdnase to the sample. Monarch rnase a is a component of the monarch genomic dna purification kit, which can be used to purify genomic dna from a variety of sample types. Available on gsava contract for federal government. Dnase i, amplification grade, digests single and doublestranded dna to oligodexyribonuleotides containing a 5 phosphate. Rna isolation with trizol invitrogen and qiagen rnaeasy this protocol applies to. Thermo scientific dnase i, rnasefree is an endonuclease that digests single and. Dnase treatment following thawing of cryopreserved pbmc is a procedure suitable for lymphocyte functional studies. Is it necessary to do a dnase treatment after every rna. The protocol mentions the possibility of adding up to 3 ul of enzyme to the mix. It has increased affinity for dnabinding and remains active in the presence of salt. It functions by hydrolyzing phosphodiester linkages, producing mono and oligonucleotides with a 5phosphate and a 3hydroxyl group.
This dnaseseq protocol was derived from methodologies originally described by boyle et al. Deoxyribonuclease i dnase i amplification grade sigmaaldrich. Dnase i, rnasefree is an endonuclease that nonspecifically cleaves dna to release di, tri and oligonucleotide. Turbo dnase is recombinant, engineered form of dnase i that is efficient than wild type dnase i in digesting away trace amounts of unwanted dna turbo dnase binds dna substrates 6fold more tightly than traditional dnase i, making enzyme tool of choice for clearing residual dna that can generate a false positive signal in rtpcr applications. Overview recombinant dnase i is an essential tool for all applications requiring dnafree rna templates. Invitrogen turbo dna free kit 50 reactions products. Dnase i and proteinase k eliminate dna from injured or. In the protocol above, dnase is inactivated by addition of edta to a final concentration of 2. For convenience, the 10 turbo dnase buffer and the dnase inactivation. Thermo scientific dnase i, rnasefree is an endonuclease that digests single and doublestranded dna.
Invitrogen dnase i, amplification grade nondistribution product as customer accommodation. For further use of the rna for expression analysis, it is highly recommended to treat the sample with dnase, an enzyme that digests dna. Dnase i definition of dnase i by the free dictionary. This formulation is purified from cow pancreas, is free of contaminating protease or dnase activity, and contains no residual host dna. The specific activity of the enzyme is 30 umg, corresponding to 1. Starrlab dnase treat rna using ambion turbo dnafree kit.
There are a number of valid protocols for dnase i treatment available to reduce cell clumping. Studies of dnaprotein interactions by dnase i, rnasefree footprinting 1. Dnase i has been purified to remove rnase activity, and is suitable for eliminating dna from rna preparations prior to sensitive applications, such as rt. Deoxyribonuclease i, amplification grade dnase i, amp grade digests. Superscript iii firststrand synthesis system for rtpcr. Clickit tunel alexa fluor imaging assay protocol fasttrack. Preparations of pbmcs can be frozen and stored at low temperatures, which allows for longterm storage and transportation. To work with larger amounts of rna, scale up the reaction including volume linearly.
Invitrogen was founded in 1987 by lyle turner, joe fernandez, and william mcconnell and was incorporated in 1989. Dnase i, amplification grade digests single and doublestranded dna to oligodexyribonuleotides. This protocol describes the preparation of and treatment with dnase i to degrade dna in solutions containing iron chloride, edtamg ascorbate buffer, and cesium chloride. It hydrolyzes phosphodiester bonds producing mono and oligodeoxyribonucleotides with 5phosphate and 3oh groups. This protocol addresses the issue of genomic dna contamination in rna samples using the ambion applied biosystems turbo dnase. A special procedure is used to remove rnases from the dnase preparation. The company initially found success with its kits for molecular cloning notably, the librarian, a kit for making cdna libraries, and the fasttrack kit. The rnasefree dnase set is intended for molecular biology applications. Dnase i, amplification grade is purified and tested for nondetectable levels of rnase contamination. If youre doing some assay that uses an rna ligase, again dnase isnt really necessary as the ligase is highly specific to rna. Clickit tunel alexa fluor imaging assay protocol fasttrack mag. Dnase i, amplification grade thermo fisher scientific. Ultrapure dnasernase free distilled water from invitrogen,ultrapure dnasernase free distilled water is designed for use in all molecular biology applications.
It is a glycoprotein of a molecular weight of approximately 39 kd. We want to remove dna contamination to the best of our abilities because our interest in environmental stresses is in the gene. Ribonuclease has been reduced to nondetectable levels. The rna can then be stored for long periods of time, at 200c. If using rna for rtpcr, treat with dnase either use dnase i protocol or turbo dnase by ambion protocol expected yields depend on cell type and tissue type, but can expect the following. Rnase a, dnase and proteasefree 10 mgml thermofisher en0531 sodium acetate buffer solution 3m, ph 5. Dnase i, amplification grade, is suitable for eliminating dna during critical rna purification procedures such as those prior to rnapcr amplification. The reaction buffer is supplied with the dnase enzyme and is required for optimal dnase activity. Dna digestion in solution protocol following rna elution.
Removal of template dna following in vitro transcription 1, see protocol on reverse page. Inactivate the dnase i by the addition of 1 l of 25 mm edta solution to the reaction mixture. Proteinase k was added in a final concentration of 0. Dnase i recombinant, rnasefree from bovine pancreas.
Dnase i from bovine pancreas is a glycoprotein of mr 37000. The protein concentration of rnase, dnasefree is 0. Deoxyribonuclease i, amplification grade thermo fisher scientific. One kunitz unit is defined as the amount of dnase i that causes an increase in a 260 of 0. Dnase i, amplification grade, is suitable for eliminating dna during critical rna. If you would like this enzyme plus reagents to inactivate the enzyme a. Dnase i is useful in nick translation for introducing singlestranded nicks that serve as primer sites for initiation of dna synthesis and for cloning random dna fragments by cleaving doublestranded dna.
787 1568 928 1265 118 798 825 1215 421 1025 498 834 912 535 1633 42 26 425 792 1143 1119 873 1031 420 111 1239 402 1024 308 92 81 970 576 1448 535 1053 68 317 575