DNase treatment

Comment It is critical to remove excess cap analogue from in vitro transcribed transcripts prior to transfection, because the cap analogue will compete with transcripts for the cellular translational machinery. Also, even after DNase treatment, the RNA sample may still contain traces of functional pDNA, which may interfere with subsequent detection by RT-PCR. Furthermore, plasmids containing a mammalian promoter may even give rise to de novo transcription in transfected cells. 

Zea mays and mutant 3 days etiolation and DNase treatment uses leaves Purer cpDNA 30... 

Fig. 31.5. Gene shuffling using a single gene (1) or a family of genes (2) (A) errorprone PCR generating diversity (B) DNAse treatment (C) homologous recombination of fragments and generation of diversity.

Although RT-PCR based methods are promising for RNA amplification, they require a DNAse step to remove DNA so that it is not coamplified along with RNA (68). Unfortunately, DNAse treatment has been reported to not be completely effective in this regard, thus additional complex steps are required (69). 

Porcine femur (cortical bone), and costa (trabecular bone) Hydrostatically pressurized at 980MPa+DNase treatment Rat bone marrow-derived mesenchymal stromal cells (MSCs) NA Hashimoto et al. (2011)... 

A DNase treatment (Nass, 1969a) also reduces contamination by nuclear DNA since generally DNase cannot enter intact mitochondria. Degradation of mt DNA by DNase may occur in damaged or leaky mitochondria. 

Once the RNA has been checked, DNase treatment is required to remove DNA that will interfere with the PCR process, giving rise to bands that do not correspond to cDNA. Resuspend part of the RNA solution (5-50 pg) in 42 pL of nuclease-free water and add 5 pL of any clean lOX restriction enzyme buffer, 2 pL of DNase I RNase-free, and 2 pL of RNase guard. Incubate at 37 C for 30 min. Store the rest of the RNA at -80 C. 

Apply the sample, including any precipitate that may have formed, to an RNeasy mini column placed in a 2-mL collection tube. With the tube closed, centrifuge for 15 sec at 17,900g. Discard the flow-through. If the volume exceeds 700 pL, load and centrifuge aliquots successively onto the RNeasy column. After the RNA has been loaded onto the Qiagen column, it is ready for DNase treatment. 

For use in microarray applications, we always include the DNase treatment step when purifying total RNA by column-based protocols. 

A similar protective action was foxmd both in the case of pre- (5 min) and postincubation (30 min) of the extract with irradiated cells. Thus a screening action of the extract can be largely excluded by these observations. Heating the dialysate for 10 min at SOX and 70 0 or for 1 min at 92°C caused some reduction of its activity, which still remained relatively high, but upon heating for 12 min at 92 C the activity was reduced three-fold. Reactivative activity of the extract was retained after storage up to 1.5 months at -20 C and more than one year after lyophilization. Lipase treatment had no effect on the reactivative activity of the extract RNAse and especially DNAse treatment lowered it significantly, while proteinase K treatment alone or followed by trypsin treatment reduced the activity 5.5-fold or completely, respectively. 

Notes Overlay PCRs with mineral oil or use a thermal cycler with a heated lid. DNase treatment is essential to degrade DNA that may contaminate commercial preparations of Taq DNA polymerase. It is convenient to perform this reaction in a PCR tube and the incubations in a thermal cycler. This recipe provides enough enzyme for five PCR samples scale up or ... 

DNAse treatment of blended tissue separation at interface of 2M sucrose layer after centrifugation Homogenization and isopycnic centrifugation... 

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