Jul 16, 2025
Polyamine Buffer for Bacterial Artificial Chromosome (BAC) DNA
This protocol is a draft, published without a DOI.
- Thom Saunders1,
- Zachary Freeman1
- 1University of Michigan
- Transgenic Animal Model Core
Protocol Citation: Thom Saunders, Zachary Freeman 2025. Polyamine Buffer for Bacterial Artificial Chromosome (BAC) DNA. protocols.io https://protocols.io/view/polyamine-buffer-for-bacterial-artificial-chromoso-g5k9by4z7
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 16, 2025
Last Modified: July 16, 2025
Protocol Integer ID: 222593
Keywords: polyamine buffer for bacterial artificial chromosome, transgenic mice from bacterial artificial chromosome, bacterial artificial chromosome, yeast artificial chromosome, artificial chromosome, large dna molecule, standard dna resuspension buffer, long dna molecules from fragmentation, polyamine buffer, long dna molecule, electron microscopy with polyamine, such long dna molecule, mb of dna, visualization of large dna molecule, kb of dna, dna abstract, polyamine, dna, generating transgenic mice, transgenes in the production, transgenic mice, transgenesis efficiency, transgenic rat, recombinase, generation of amelx, transgene, icre mice supports ameloblast, optogenetic application to dopamine, optogenetic application
Abstract
Abstract
Bacterial Artificial Chromosomes (BAC) can include over 300 kb of DNA while Yeast Artificial Chromosomes (YAC) can include as much as 1 Mb of DNA. Such long DNA molecules are prone to fragmentation when stored in standard DNA resuspension buffers such as 10 mM Tris, 1 mM EDTA (Montoliu et al. 1995, Van Keuren et al. 2009). To protect long DNA molecules from fragmentation so that they can be used as templates for recombineering (Zeidler and Saunders, 2019) or as transgenes in the production of transgenic mice (Said et al. 2019) and transgenic rats (Witten et al. 2011).
Montoliu L, Bock CT, Schütz G, Zentgraf H. 1995. Visualization of large DNA molecules by electron microscopy with polyamines: application to the analysis of yeast endogenous and artificial chromosomes. J Mol Biol. 246:486-92.
Said R, Zheng L, Saunders T, Zeidler M, Papagerakis S, Papagerakis P. 2019. Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1. J Dent Res. 98:1002-1010.
Van Keuren ML, Gavrilina GB, Filipiak WE, Zeidler MG, Saunders TL. 2009. Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Res. 18:769-85.
Witten IB, Steinberg EE, Lee SY, Davidson TJ, Zalocusky KA, Brodsky M, Yizhar O, Cho SL, Gong S, Ramakrishnan C, Stuber GD, Tye KM, Janak PH, Deisseroth K. 2011. Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement. Neuron. 72:721-33.
Materials
Reagent | Source | Cat. No. | Stock Concentration | Final Concentration
--- | --- | --- | --- | ---
Tris-HCl, pH 7.4 | SigmaAldrich | 648315 | 100 mM | 10.0 mM
EDTA | SigmaAldrich | E7889 | 500 mM | 0.01 mM
Spermine Tetrahydrochloride | SigmaAldrich | S-1141 (1 g) | See polyamine stock | 30 microM
Spermidine Trihydrocholoride | SigmaAldrich | S-2501 (1 g) | See polyamine stock | 70 microM
NaCl | SigmaAldrich | S5150 | 5 M | 100 mM
Water for DNA | SigmaAldrich | W4502 | |
Sterile 0.22 micron filter | | | |
Sterile tubes | Various sizes | | |
Troubleshooting
Safety warnings
Store DNA at 4°C. DO NOT STORE RESUSPENDED DNA IN THE FREEZER.
Procedure
| A | B | C | D | E | |
| Reagent | Source | Cat. No. | Stock Concentration | Final Concentration | |
| Tris-HCl, pH 7.4 | SigmaAldrich | 93313 | 1 M | 0.01 M | |
| EDTA | SigmaAldrich | E7889 | 500 mM | 0.01 mM | |
| Spermine Tetrahydrochloride | SigmaAldrich | S-1141 (1g) | See 1000X polyamine stock | 30 uM | |
| Spermidine Trihydrochloride | SigmaAldrich | S-2501 (5g) | See 1000X polyamine stock | 70 uM | |
| NaCl | SigmaAldrich | S5150 | 5 M | 100 uM | |
| Water for DNA | SigmaAldrich | W4502 | |||
| Sterile 0.22 micron filters | |||||
| Sterile tubes | various sizes | ||||
List of Materials for polyamine buffer
Prepare 1000X polyamine stock. Polyamine powders are extremely hygroscopic. It is recommended that small quantities be ordered and used entirely in the preparation of a 1000X Polyamine stock solution. Dissolve 0.94 g spermine and 3 g spermidine together in 123.0 ml sterile water for DNA. Then filter sterilize through 0.22 micrometer filtration. Prepare aliquots and store at -20°C.
Prepare Polyamine Microinjection Buffer
| A | B | |
| Solution | Volume | |
| 1 M Tris-HCl, pH7.4 | 0.5 ml | |
| 0.5 M EDTA | 0.01 ml | |
| 5 M NaCl | 1.0 ml | |
| 1000X Polyamine Stock | 0.05 ml | |
| Water for DNA | 48.44 ml | |
Resuspend DNA in polyamine buffer and quantitate with your preferred method. It may be necessary to allow DNA pellets to enter solution during an overnight incubation in the refrigerator. Store DNA at 4°C. DO NOT STORE RESUSPENDED DNA IN THE FREEZER.