By Abelson J. (ed.), Simon M. (ed.), Wu R. (ed.)
The looks of one other quantity in that first-class sequence, tools in Enzymology, is usually a reason for appreciation should you desire to effectively perform a specific process or organize an enzyme or metabolic intermediate with out the tiresome prospect of looking through strange literature and maybe picking an unproven process which isn't simply reproduced.
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Additional resources for Recombinant DNA Part G
Resuspend the bacterial pellet thoroughly by vortexing in 200/xl of TE buffer. Place in a boiling water bath for 1 min. 2~ 2. 5 M NaCI]. Vortex for 15 sec. Add 400/zl of a 1 : 1 phenol-chloroform mixture. 23 Vortex vigorously for 15 sec. Spin at full speed in a microfuge for 15 min at room temperature. 3. 5-ml microfuge tube. Add an equal volume of chloroform, vortex for 10 sec, and spin at room temperature for 30 sec to separate the two layers. 5-ml microfuge tube. Precipitate the plasmid DNA by mixing with an equal volume of ice-cold 2-propanol.
Spin at full speed for 5 min in a microfuge at room temperature to separate the aqueous and organic layers. 5-ml microfuge tube, and extract with 400/~1 of chloroform. Spin at full speed for a few seconds in a microfuge at room temperature. 5-ml microfuge tube. Precipitate the nucleic acids by adding 800 /~1 ethanol and mixing well. Store for 30 min at - 7 0 °. Spin down for 15 min at full speed in a microfuge at 4°. 9. Dissolve the DNA pellet in 20/zl of TE [TE: 10 mM Tris-HCl (pH 17 Please note that the uncut plasmids tested here will exhibit multiple bands corresponding to different topological forms.
MI3 DNA was isolated as in protocol 3. Sequencing reactions were performed using the KiloBase Sequencing System (Bethesda Research Laboratories) according to the manufacturer's instructions. Reaction products were resolved on a 6% polyacrylamide-8 M urea gel at 60 W in Tris-borate EDTA buffer. The first four lanes (left-hand side) are reactions using DNA isolated by standard phenol extraction procedures (Sambrook e t a l . ) . 4 The other four lanes (right-hand side) are reactions using DNA captured and released from the capture reagent.