Navigation Links
Electroporation of Yeast Artificial Chromosomes

Maren Bell and Robert Mortimer, Human Genome Center, Division of Cell and Molecular Biology, Lawrence Berkeley Laboratory, Berkeley, California 94720


Introduction
Transformation of yeast with yeast artificial chromosomes (YACs) has traditionally been performed by a PEG-spheroplast procedure.1,2 However, the procedure is complicated, often unreliable, and transformation efficiencies are relatively low when compared to more recently developed transformation methods. An additional concern is that the smaller YACs of any given ligation mixture are cloned preferentially, therefore prior size fractionation with sucrose gradients to enrich for larger molecules, or treatment with spermidine to improve transformation efficiency of larger molecules, is required to obtain reasonable efficiencies with larger YACs. Several electroporation protocols have recently been reported for the transformation of Saccharomyces cerevisiae with supercoiled plasmids.3-6 In an attempt to improve the currently used YAC cloning procedure, we investigated the electroporation of S. cerevisiae strain AB1380 (Mat a, ade2-1, can1-100, lys2-1, trp1, ura3, his5, psi + ) with a 14.4 kb mini-YAC. Initial experiments showed transformation efficiencies two-fold better than those reported in a previously published YAC electroporation protocol.7


Materials and Methods
The YAC used in our experiments was constructed by the insertion of a 4.6 kb lambda MluI fragment into the MluI-SUP4 cloning site of pYAC-RC. 8 E. coli was transformed by the resulting 16.1 kb plasmid, the DNA was isolated, purified over cesium chloride, and restriction d igested with BamHI, thereby freeing the telomeres and resulting in a 14.4 kb linear DNA molecule. The restriction-digested DNA was microdialized (Millipore VS, 0.025 m) against TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) for 20 minutes, ethanol precipitated, and suspended in TE. The electroporation protocol used for our experiments was an adaptation of the one devised by Becker and Guarente.5 Exponentially growing cells were harvested by centrifugation, washed twice in water (4 C), once in 1 M sorbitol (4 C), and finally resuspended in a 2/3 pellet volume of 1 M sorbitol (4 C). A 40 l aliquot of cells was gently mixed with 1-5 l of DNA (0.1 g) just before electroporation. A Gene Pulser apparatus connected to the Pulse Controller accessory was used to pulse the sample in a chilled 0.2 cm cuvette, generating a pulse with a field strength of 7.5 kV/cm (1.5 kV), and a time constant (τ) of approximately 4.2 msec (25 F and 200 Ω). Ice cold 1 M sorbitol (1 ml) was added immediately after pulse delivery, and 150 l aliquots were plated on SD ura- plates. Transformants were visible after 3 days of incubation at 30 C.


Results and Discussion
Transformations with the small YAC have yielded approximately 700 transformants / g of DNA. Transformations with a supercoiled 5 kb plasmid yielded up to 1.6 x 104 transformants / g of DNA. A number of parameters were investigated in attempt to increase efficiencies. We found that 0.1 g of DNA in 1-5 l of TE or water was the optimum amount for 40 l of cells (5 x 108 cells). More DNA had an adverse effect on transformation efficiency and less DNA did not seem to saturate the reaction (Figure 3). Spermidine -treated DNA did not yield any transformants. Exponentially growing cells gave the most consistent and highest transformation efficiencies.

A possible problem associated with the high voltage electroporation of very large DNA fragments such as YACs is that the molecules are most likely sheared before they have a chance to enter the cell. Lower voltage experiments with the Gene Pulser apparatus (which generates an exponential decay pulse) have a less disruptive effect on large DNA molecules, but they also yield lower transformation efficiencies.3,4 The use of alternative waveforms may improve on current results.6 The S. cerevisiae strain used here (AB1380) is traditionally employed in PEG-spheroplast transformations and we do not know whether this strain is also a good electroporation candidate. Other strains that have proven to yield high transformation rates might further improve the results presented here.


References
1. Burke, D. T., Carle, G. F. and Olson, M. V., Science, 236, 806-812 (1987).

2. Burgers, P. M. J. and Percival, K. J., Anal. Biochem., 163, 391-397 (1987).

3. Delorme, E., Appl. Environ. Microbiol., 55, 2242-2246 (1989).

4. Simon, J. R. and McEntee, K., Biochem. Biophys. Res. Comm., 164, 1157-1164 (1990).

5. Becker, D. M. and Guarente, L., Meth. Enzymol., 194, 182- 186 (1990).

6. Meilhoc, E., Masson, J.-M. and Teissi, J., Bio/Technol., 8, 223-227 (1990).

7. Rech, E. L., Dobson, M. J., Davey, M. R. and Mulligan, B. J., Nucl. Acids Res., 18, 1313 (1990).

8. Marchuck, D., and Collins, F. S., Nucl. Acids Res., 16, 7743 (1988).


Acknowledgements
This work was supported by Bio-Rad Laboratories and by funds administered through DOE Contract No. DE-ACO3- 765F00098 to the Human Genome Center at Lawrence Berkeley Laboratory.


back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
2. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
3. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
4. Optimizing Chemical Transfection and Electroporation of siRNAs
5. High Throughput siRNA Electroporation
6. Electroporation of Primary Bone Marrow Cells
7. Production of Hybridomas by Electroporation
8. Transformation of Filamentous Fungi by High-Voltage Electroporation
9. Transgenic Zebrafish by Electroporation
10. Transient Gene Expression Following Single and Double Exponential Decay Electroporation Pulses
11. Epitope-Tagging Vectors for Functional Analysis in Yeast
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:5/25/2016)... FL (PRWEB) , ... May 25, 2016 , ... Lady ... eight she tore her cruciate ligament in her left knee. Lady’s owner Hannah sought ... a central Florida board-certified veterinary surgeon, to repair her cruciate ligament and help with ...
(Date:5/24/2016)... ... May 24, 2016 , ... Cell ... injuries, will be accelerated by research at Worcester Polytechnic Institute (WPI) that yielded ... healing and tissue regeneration. , The novel method, developed by WPI faculty members ...
(Date:5/24/2016)... ... 24, 2016 , ... Last week, Callan Capital, an integrated wealth management firm ... Future of San Diego Life Science event at the Estancia La Jolla Resort and ... event with speakers Dr. Rich Heyman, former CEO of Aragon and Seragon, and Faheem ...
(Date:5/23/2016)... , ... May 23, 2016 , ... The need for blood donations in South Texas ... by the South Texas Blood & Tissue Center, blood donations are on the decline. In ... and they are down 21 percent in South Texas in the last four years alone. ...
Breaking Biology Technology:
(Date:3/17/2016)... March 17, 2016 ABI Research, the ... the global biometrics market will reach more than ... increase from 2015. Consumer electronics, particularly smartphones, continue ... sensors anticipated to reach two billion shipments by ... Dimitrios Pavlakis , Research Analyst at ABI Research. ...
(Date:3/14/2016)... NXTD ) ("NXT-ID" or the "Company"), a company ... of a new series of commercials on Time Warner Cable ... .  The commercials will air on Bloomberg TV, Fox Business ... show. --> NXTD ) ("NXT-ID" or the "Company"), ... the airing of a new series of commercials on Time ...
(Date:3/11/2016)... --> --> ... Recognition Market by Technology (Pattern Recognition), by Component (Hardware, ... Type (On-Premises and Cloud), by Industry Vertical and by ... the global market is expected to grow from USD ... 2020, at a CAGR of 19.1%. , ...
Breaking Biology News(10 mins):