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5 volumes of ethanol 6. 7. 8. 9. ) and air-dry Resuspend in 4 µl LoTE Use 1 µl ligation mix to transform ELECTROMAX DH10B cells Any transformation procedure can be used, but we recommend electroporation due to its high transformation efficiency in combination with the extremely competent ELECTROMAX DH10B cells (> 1010 transformants/µg plasmid). 1. Allow a vial of 100 µl ELECTROMAX DH10B cells to thaw on ice 2. Meanwhile chill the required number of electroporation cuvettes on ice 3. When thawed, proceed immediately with the ELECTROMAX DH10B cells.

12. 13. 14. 15. 16. 17. 000 rpm Wash pellet 1x with 70% ethanol, then allow to air-dry Resuspend in 30 µl LoTE Load entire sample on 8 lanes of a 12 % polyacrylamide gel. Use a 10-bp ladder as marker. 5 hrs or until Orange G dye is at the bottom of the gel Stain gel with ethidium bromide Excise the ditag band from gel. 5 ml PCR tubes that have been pierced with a 21-gauge needle. 000 rpm to fragment the polyacrylamide. 5 ml PCR tubes and add 300 µl LoTE to each tube. Vortex and then incubate at 37°C for 15–30 min Fig.

PCR Methods Appl 4:S97–108 Chomczynski P Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 162:156–159 Chomczynski P, Mackey K, Drews R Wilfinger W (1997) DNAzol: a reagent for the rapid isolation of genomic DNA. Biotechniques 22:550–553 Duboule D (1997) The evolution of genomics [editorial]. Science 278:555 Ghosh S, Karanjawala ZE, Hauser ER, Ally D, Knapp JI, Rayman JB, Musick A, Tannenbaum J, Te C, Shapiro S, Eldridge W, Musick T, Martin C, Smith JR, Carpten JD, Brownstein MJ, Powell JI, Whiten R, Chines P, Nylund SJ, Magnuson VL, Boehnke M Collins FS (1997) Methods for precise sizing, automated binning of alleles, and reduction of error rates in large-scale genotyping using fluorescently labelled dinucleotide markers.

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Modern Neuroscience Research Protocol


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