RNase-free DNase

A Working Solution of the stain should be prepared just before use, by combining 10 ml of 0.1% Triton X-100 in phosphate-buffered saline, 2 mg RNase (DNase-free, Sigma), and 200 pi PI solution (Sigma, 1 mg/ml in distilled water). Stock solutions of Triton X-100 and PI should be stored at 4°C. Centrifuge the ethanol-fixed cells (200 x g for 5 min) and discard the supernatant, removing it completely. 

Apply the following proportion to calculate the volume of RNase-DNase-free H20 to add to the primer ... 

Spin down the tube containing the lyophilized primers in order to avoid the dispersion at the time of opening. Add RNase-DNase-free water. Equilibrate at room temperature for 5-10 min before use (see Note 4). 

Panomics TranSignaF TF-TF Interaction Array Membranes and Hybridization Reagents (Box 1) containing TranSignaF TF-TF Interaction Arrays, Hybridization Buffer, Substrate Solution I, Substrate Solution II, Substrate Solution III, lOOOx Streptavidin HRP Conjugate, 20x SSC, 20%SDS, 4x Wash Buffer, lOx Detection Buffer, Distilled HjO (RNase, DNase-free). 

Briefly air dry the pellet and resuspend the RNA in an appropriate volume of ice-cold RNAse- Dnase-free water. Allow the peUet to sit in the water for at least lOmin on ice before resuspending by pipeling. If the pellet does not dissolve fully, incubate at 37°C for 5 min. It is important that the final concentration is sufficiently high to continue with the labeling procedures. Using this method, it is not necessary to add RNase inhibitors to ensure viability. 

RNase A (DNase-free) stock solution of 1000 U diluted in 1 mL PBSG. 

All chemicals were RNase and DNase free (Sigma, Italy). All water and pipette tips were sterilised by autoclaving for 30 min (120°C). 

Phenolic extraction of cell lysates is one of the oldest techniques in DNA preparation. Examples of these have been presented in Chapters 6 and 7. Single cells in suspension are lysed with a detergent, and a proteinase enzyme is used to break down the protein molecules. Non-nucleic acid components are then extracted into an organic (phenol-chloroform) solvent, leaving nucleic acids in the aqueous layer. Two volumes of isopropanol are added to the isolated aqueous phase to precipitate the high-molecular-weight nucleic acids as a white mass. These are then treated with DNase-free ribonuclease (RNase) to remove the RNA. This is followed by a second treatment with proteinase, phenol extraction, and isopropanol precipitation. After precipitation, the DNA is separated from the isopropanol by... 

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

Propidium iodide (PI) staining solution PBS + 5 pg/mL PI (Molecular Probes, Eugene, OK) + 200 pg/mL DNase-free RNase (Sigma) make up fresh immediately before use. The RNase can be made as a stock of 20 mg/mL in H20 and stored in aliquots at -20°C. 

X Tris/borate/EDTA, pH 8.3 DNase-free, RNase-free, and protease-free buffer. 

Stock solution of RNase Dissolve 2 mg of DNase-free RNase A (Sigma Chemical Co., St Louis, MO) in 1 mL of distilled water. If DNase-free RNase is unavailable, DNase activity is destroyed by boiling the stock RNase solution for 5 min. Aliquots can be stored at -20°C. 

It should be noted that sterility is very important when performing the nuclear extraction and first few steps of the TF protocol. Make sure that the water that is being used is RNase-free, DNase-free, and sterile. All centrifuge tubes must be sterile or previously autoclaved. All pipeting and transferring of materials should take place under a sterile hood. The derived nuclear extract is very sensitive to post extraction modifications and should therefore be handled very carefully. Once a sample is extracted and the protein concentration is taken, the sample should be used immediately or else aliquoted and frozen at -80 °C. Also, in order to assure consistency of results between trials, it is beneficial to use aliquots from the same original batch. 

Treatment with RNase A removes contaminating RNA and this can either be integrated into the purification procedure or performed after the DNA has been purified. Prior to use, ensure that the RNase A solution has been heat-treated to destroy any contaminating DNase. Alternatively, use DNase-free RNase purchased from a reliable supplier. 

Add DNase-free RNase A to a concentration of 50 /tg/ml and incubate at 37°C for 30 min. Add SDS to a final concentration of 0.25% and proteinase K to 250 ju,g/ml. Incubate at 37 C overnight. Incubate further for 6 hr at 68°C. Extract samples two to three times with phenol/chloroform, and once with chloroform. Ethanol precipitate DNA in the presence of 20 /ig glycogen at -20 C overnight. 

Add an appropriate vol. of 10 mM Tris-HQ buffer pH 8.0 containing 1 mM EDTA and 20 ug/ml of DNase-free pancreatic RNase. 

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