DNA preparation by pulsed-field gel electrophoresis (isolation of yeast intact DNA)
To prepare yeast DNA for electrophoresis, the cell walls of yeast cells are enzymatically disrupted and then suspended in melted low melting point agarose to ma
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DNA preparation by pulsed-field gel electrophoresis (isolation of mammalian cell and tissue DNA)
This protocol describes methods for preparing DNA samples from cultured cells and tissues. Leukocytes of patient or animal origin can also be used as high molec
Recovery of DNA fragments from polyacrylamide gels (crush and soak method)
The standard method for recovering DNA from polyacrylamide gels is the "crush and soak" technique first introduced by Maxam and Gilbert (1977). The eluted DNA i
Restriction endonuclease digestion of DNA in agarose gel plugs
Chromosomal DNA isolated from mammalian cells, yeast or bacteria can be digested in agarose plugs with the desired endonuclease. The low melting point agarose p
The cloned PCR product is ligated into a T vector.
DNA fragments amplified by Taq DNA polymerase PCR with a 3' prominent A base are efficiently cloned into T vectors that have an unpaired 3' T base complementary
Southern hybridization of radiolabeled probes with membrane-immobilized nucleic acids
The signal intensity obtained by Southern hybridization depends on several factors, including the ratio of immobilized DNA complementary to the probe, the size
Oligonucleotides and excess dNTP were removed from DNA amplification products by ultrafiltration.
At the end of the PCR reaction, it is essential to remove oligonucleotide primers, primer dimers and dNTP from the amplified DNA product. This experiment is bas
Radiation autoradiography of DNA in polyacrylamide gels
Radioactive DNA bands separated by acrylamide gel electrophoresis can be detected by radioautography. Polyacrylamide analytical gels containing radioactive DNA
Anchoring enzyme digestion of cDNA
If the previous step in the series of analytical techniques for gene expression, SAGE cDNA, was stored at -20°C, then this experiment was thawed slowly on ice.