Recovery of DNA from agarose gels (DEAE-cellulose membrane electrophoresis)
Recovery of DNA from agarose gels can be applied to (1) mammalian cell transformation and (2) radiolabeling.
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Hybridization experiments with oligonucleotide probes in solution
Jabobs et al. (1988) described the method and principles of hybridization in quaternary ammonium-containing buffers. The following is a simple variation of this
Direct cDNA screening by large fragment genomic DNA cloning
This protocol uses a 500kb contiguous sequence of human genomic DNA cloned in a BAC vector to directly screen for cDNAs complementary to it, but the method can
Experimental Determination of Unwinding Temperature
The unlinking temperature for hybridization of oligonucleotides to target sequences can be calculated using the method described in the overview of this chapter
Experiments with oligonucleotides synthesized by labeling with Escherichia coli Klenow fragments
Oligonucleotides are usually labeled using a phosphorylation reaction catalyzed by the T4 phage polynucleotide kinase. However, there is sometimes a need to lab
Eukaryotic expression library construction and screening experiments
Similar to Scheme 1, Scheme 2 describes the method of cDNA library construction and screening on eukaryotic expression vectors, and the process is divided into
Rapid isolation of mammalian DNA
Mammalian DNA prepared according to this protocol is approximately 20-50 kb and is suitable for use as a template for PCR reactions. the DNA yield varies betwee
Purification of RNA from tissues and cells by acid phenol-guanidine thiocyanate-chloroform extraction
The cellular RNAase should be inactivated as soon as possible during the first stage of the extraction process. Once the endogenous RNAase is destroyed, the pos
Extraction of poly(A) + RNA by oligo(dT)-cellulose chromatography
In contrast to rRNA, 5SRNA, 5.8 SRNA and tRNA, most eukaryotic mRNAs have a poly(A) tail at their 3' end. Therefore, mRNA can be isolated from total cellular RN