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
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Recovery of DNA from agarose and polyacrylamide gels (electroelution to dialysis bags)
This technique (McDonell et al., 1977) allows the recovery of double-stranded DNA from agarose or polyacrylamide gel slices over a wide range of molecular masse
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
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.
Experiments on purification of radiolabeled oligonucleotides by Sep-Pak C18 column chromatography
This scheme describes a method to separate radiolabeled oligonucleotides from undoped radiolabel using the property of antiphase affinity of oligonucleotides fo
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
Pulsed-field gel electrophoresis with clamped uniform electric field
In clamped homogeneous electric field (CHEF) gel electrophoresis, the electric field is generated by multiple electrodes. These electrodes are arranged in a qua
Molecular quality criteria for pulsed-field gel electrophoresis
PFGE is used to isolate very large DNA molecules and thus requires standards of very high molecular mass. Such standards can be obtained from phages such as T7
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