DNA carriers

Gietz, R. D., and Woods, R. A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87-96. 

Dilute 4 to 5 (ig of DNA into 0.1 ml of 250mM CaCb, Carrier DNA such as salmon sperm or empty plasmid can be used to make up the amount if the DNA is less than 4 to 5 jxg. 

The cells were transfected with 50 ng of HaloTag expression plasmid with 150 ng of pcDNA-DEST47 GFP as a carrier DNA using FuGENE6. 

Carrier DNA. Sonicated salmon sperm DNA, 10 mg/mL m double-distilled water. 

Excess carrier DNA may be added at this state (see Note 7)... 

Carrier DNA (e g., salmon sperm) can be added to the reaction mix m excess to reduce nonspecific probe binding. I have not found this necessary for the detection of human papillomaviruses m archival surgical biopsies, but it is often necessary for cultured cells and cervical smears (14). 

For each mutant, a sample containing the pCLI plasmid of interest and carrier DNA (e.g., Salmon testes DNA, Sigma) in a total of 5 fig of DNA in less than 10 fil volume is added to 100 pi of competent cells placed in the bottom of a sterile 12-ml Falcon 2051 tube (Becton Dickinson, Lincoln Park, NJ). The contents are swirled gently to mix, and the tube is incubated for 10 min. 

Add 50 ng 32P-labeled DNA probe to carrier DNA and heat to 100°C for 10 min. Add to 10 mL hybridization mix (3X SSC, 2% SDS, 5X Denhardf s, 0.2 mg/mL sheared carrier DNA). Remove prehybridization solution and replace with hybridization solution containing probe. Incubate in hybridization oven or shaker overnight at 65°C. 

Carrier DNA should be sheared with a sonicator to decrease the viscosity. Large quantities can be prepared at a time and a sonicator is needed infrequently. Alternatively, sheared carrier DNA can be purchased for a minimal extra charge. 

As for dot blot hybridization, RNA probes can be used to detect RNA targets but these probes could not be stripped from the membrane due to the higher stability of RNA RNA hybrids (Srivastava and Schonfeld, 1991). Moreover, RNA probes make very stringent washes mandatory (e.g., (pre)hybridization at 60°C and in 5 X SSPE, 50% formamide, 0.2% SDS, 200 xg/ml denatured carrier DNA and 200 (ig/ml yeast tRNA and washes, after a hot rinse , in 0.1 X SSPE, 0.1% SDS at 60°C) and may still lead to spurious binding of probes to rRNA. Normalization of blots by rehybridizing with a probe to a cellular function suffers from the variation in the level of expression of some genes. Instead, jS-actin or 28S rRNA probes (Barbu and Dautry, 1989) are suggested. 

The hybridization buffer usually contains 5 X SSC, 50% formamide, 100 xg/ml carrier DNA/tRNA, 0.1% Triton X-100 and 20 mM vanadyl ribonucleoside in addition to denatured probe. In the case of optimal probes of 150 bases (40-60% GC), the optimal hybridization temperature (T — 25 0 in this buffer would be about 56-70°C for RNA probes and 37-6 TC for DNA probes (eqs. (11) and (12), Chapter 2). However, very high temperatures, e.g., > 50°C, affect tissue quality. Usually, incubation is at 20-50°C in a humidified environment. 

Add 240 pi of 50% PEG-3350, 36 pi 1.0 M LiAc, 50 pi of herring testes carrier DNA (2 mg/ml) x pi library plasmid DNA (0.1 to 10 pi), and sterile water to a final volume of 360 pi. It is important that the components are added in the exact sequence listed above. Vortex vigorously until the cell pellet has been completely mixed. This usually takes about 1 min. Incubate at 30°C for 30 min. 

---