DNase stock

DNase stock. Add 10 mL water to 10 mg DNase. Mix well and filter sterilize. Aliquot in sterile tubes (0.5 mL/aliquot) and store at -20°C. 

Cut the dissected tissue of interest into small pieces (see Note 1). If the neural tissue is obtained from rodents from embryonic day (E) 12 up to postnatal day (P) 7, proceed directly to step 6. If the neural tissue is obtained from rodents afrer P7, or from tumors, proceed with enzymatic digestion of the tissues. To this end, dissolve 400 U of papain, 5 mg of cysteine and 5 mg of EDTA into 25 mL of EBSS. Vortex until the solution is clear, add 0.5 mL of a 0.1% DNAse stock solution and filter sterilize. 

Digest mix for one calf brain homogenate is freshly prepared by mixing 2 ml of Collagenase stock, 2 ml Trypsin stock, and 1ml DNAse stock in 15 ml of DMEMh- S. 

Add 80 pL of DNase solution. DNase solution contains 27 Kunitz U resuspended in RDD buffer. It is made by adding 10 pL of DNase stock solution (750 Kunitz U/mL ) to 70 pL RDD buffer. 

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. 

Make 50 pM stock solntions of each of the primers (assnming that 25tiM of the primers are dilnted in 500 pL of nltrapnre DNAse-/RNAse-free water, concentration of the stock primer wonld be 50 pM)-... 

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

Check the stock of reagents for reverse transcriptase and PCR reaction, RNase-free DNase, Kim wipes, parafilms, plastic coverslips. 

Fig. 2.4. Vector characterization. (A). SDS acrylamide gel electrophoresis of a rAAV-UF-11 vector. Approximately 2.5 x 1011 drp were loaded on a 10% SDS-polyacrylamide gel. The three capsid protein bands in the correct stoichiometry are visualized by silver staining. (B) Infectious center assay of a rAAV-CB-hAAT stock when probed for the transgene each spot represents a vector infected cell. (C) Dot blot analysis of the rAAV stock to determine the titer of DNase-resistant particles of rAAV. Amounts (ng) refer to a serial dilution of the packaged rAAV vector plasmid used to construct the standard curve. The stock has a calculated titer of 4.3 x 1013 drp/ml.

Metabolization of foreign substances Sinusendothelial cells are also capable of endocytosis and, by virtue of their extensive enzyme equipment, in a perfect position to metabolize different kinds of foreign substances. They are endowed with a good stock of acid phosphatase, acid DNase, p-N-acetylglucosaminidase, P-glucu-ronidase, arylsulphatase (A, B), cholesterol esterase, col-lagenases, etc. 

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. 

DNAse 1 (Worthington, Cat. LS002138) is prepared in a 3,400U/ml stock solntion in PBS, sterilized over 0.2- im filter, and stored at -20°C. 

DNase I stock solutions are stored at -2O C (1 mg/ml) in 5- xl aliquots (each aliquot is used once). Variation in the activity of DNase I preparations is often observed and the exact amount needed to introduce the desired number of nicks should be determined for each enzyme batch. Sometimes template switches occur which will result in snap-back structures (zero-binding nucleic acid), which remain S, nuclease-resistant upon denaturation. Rigby et al. (1977) suggested that this effect was due to a differential loss of 5 - 3 exonuclease activity upon storage leading to a displacement of the nicked strand and a template switch from the complementary... 

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. 

Tissue Preparation 1. P2 Sprague-Dawley Rat pups. 2. Dulbecco Modified Eagle s Medium (DMEM), high glucose, no glutamine, no pyruvate (Cambrex). 3. Ca +-Mg free Hanks Balanced Salt Solution (Cambrex). 4. 2.5% trypsin solution (Cambrex) diluted 1 10 in HBSS for 0.25% final, aliquot and store at -20°C, stable for 1 year. 5. DNAse 1 (Boehringer Mannheim) prepare 1 mg/mL stock in PBS aliquot store at -20°C stable for 1 year. 6. Heat-inactivated FBS (Cambrex) aliquot and store at -20°C. 

Unlabeled dNTPs (whichever were not included as labelled dNTP) to final concentration of 20 mM Add 1 ul diluted DNase 1 (10 ng/ml = 1 10,000 dilution of 1 mg/ml stock)... 

---