Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT)

GE Truett, P Heeger, RL Mynatt, AA Truett… - …, 2000 - Future Science
GE Truett, P Heeger, RL Mynatt, AA Truett, JA Walker, ML Warman
Biotechniques, 2000Future Science
Preparation of mouse genomic DNA for PCR is costly and laborious, primarily because of
the difficulty in physically separating DNA from other tissue components. This level of
purification is unnecessary for many PCR applications. A simple alternative, lysis in an
alkaline reagent and neutralization with a suitable buffer, has been used to prepare genomic
DNA from buccal swabs, whole blood, semen and forensic samples collected from humans
(2). A few modifications of this protocol make it possible to prepare PCR-quality mouse …
Preparation of mouse genomic DNA for PCR is costly and laborious, primarily because of the difficulty in physically separating DNA from other tissue components. This level of purification is unnecessary for many PCR applications. A simple alternative, lysis in an alkaline reagent and neutralization with a suitable buffer, has been used to prepare genomic DNA from buccal swabs, whole blood, semen and forensic samples collected from humans (2). A few modifications of this protocol make it possible to prepare PCR-quality mouse genomic DNA with a brief incubation in hot sodium hydroxide and pH adjustment with a Tris solution (HotSHOT). The Hot-SHOT method is rapid, inexpensive and may be carried out in 96-well plates, making it amenable to automation and high-throughput genotyping. The reagents for HotSHOT DNA preparation are simple to prepare. An alkaline lysis reagent with 25 mM NaOH, 0.2 mM disodium EDTA and a pH of 12 is prepared by dissolving the salts in water without adjusting the pH. A neutralizing reagent with 40 mM Tris-HCl and a pH of 5 is prepared by dissolving Tris-HCl (not Tris base) in water without adjusting the pH. Tissue samples (neonatal mouse toes, ear punches, 0.2-cm tail snips or 25-mg pieces of spleen) are collected into a 96-well thermal cycler plate or thermal cycler strip tubes. The tissue sample must be small; too large a sample can cause the method to fail. Alkaline lysis reagent (75 µL is added to the samples and heated to 95 C for 10 min to 1 h. The undissolved tissue does not interfere with PCR. After heating, samples are cooled to 4 C, and 75 µL neutralizing reagent are added to each sample. One to five microliters of the final preparation are used per each 10-µL PCR volume. The combination of the alkaline lysis and neutralizing reagents yields a buffer consisting of 20 mM Tris-HCl (pH 8.1) and 0.1 mM EDTA, which is similar to a common DNA storage buffer.
We have used this technique to prepare DNA for hundreds of PCR assays and find it remarkably consistent. Based on our initial success, we tested the method with tissues commonly used as a source of mouse genomic DNA. Toes from neonatal mice, 0.2-cm tail snips from weanling mice, ear punches from adult mice and small pieces of spleen (about 25 mg) from adult mice were placed in thermal cycler strip tubes with 75 µL alkaline lysis reagent added to each sample. Samples were heated to 95 C for 10, 20, 30 or 60 min, chilled to 4 C and 75 µL neutralizing reagent were added to each tube. The yield of soluble DNA was measured by a fluorometric assay (1). The performance of HotSHOT DNA in PCR was tested by amplifying a 182-bp PCR product and comparing the performance of DNA prepared by proteinase K digestion, followed by phenol: chloroform extraction and ethanol precipitation (3). In this experiment, 1.5 µL HotSHOT DNA was combined with 8.5 µL PCR mixture. Forward and reverse primers were combined at 200 nM each with PCR buffer, 3 mM MgCl2, 0.2 mM each dATP, dCTP,
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