What is the ramp rate for the Applied Biosystems® StepOne™ instrument?

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The ramp rates for the StepOne™ instrument are as follows:
Standard Mode Ramp Rate: sample ramp rate 1.6oC/sec up and 1.6oC/sec down
Fast Mode Ramp Rate: sample ramp rate up at 2.2oC/sec, down at 2.2oC/sec.

Answer Id: 2741

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What are some of the common types of auxotrophic markers in yeast?

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The following are commonly employed auxotrophic markers:

1) his3Δ1: Histidine requiring strain (from gene disruption) with a deletion in locus 1. The his3 denotes the disruption of the HIS3 gene. The Δ1 is a deletion that has been engineered to decrease the recombination between the incoming plasmid DNA and the chromosomal site.
2) leu2: Leucine requiring strain due to the disruption of the LEU2 gene.
3) trp1-289: Tryptophan requiring strain, developed from gene disruption and a further point mutation to decrease the recombination between the incoming plasmid DNA and the chromosomal site.
4) ura3-52: Uracil requiring.

For more detail on types and methods of gene disruption in yeast refer to METHODS IN ENZYMOLOGY Vol. 194.

Answer Id: 3770

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7f6caf1f0ba788cd7953d817724c2b6e_FAQ

What are the expression levels observed from pYES2?

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The following relative expression levels were observed in beta-galactosidase expression assays:
0.1 units when repressed in the presence of glucose.
Greater than 2000 units when induced in the absence of glucose and the presence of galactose.

Answer Id: 3772

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83ddfbd1c4f871159c148d7a010e69be_FAQ

Does the 2μ origin contain genes 1, 2, and 3 or just 3?

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The 2μ sequence in pYES2 has the replication origin only--not sequences that code for the proteins required for self replication. Therefore, vectors such as pYES2 will replicate to high copy number (30 per cell) and are very stable in circle-plus (have endogenous 2μ) strains but are maintained only at low copy number (2 per cell) and are very unstable in circle-zero strains, with no endogenous 2μ.

Answer Id: 3773

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5c151c2a9b76f9ef26d7e0f0d00c9a89_FAQ

Where is the polyadenylation sequence in pYES2?

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The polyadenylation occurs at base 103.

Answer Id: 3774

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73f104c9fba50050eea11d9d075247cc_FAQ

Does pYES2 have a CEN sequence?

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pYES2 does not have a CEN sequence. pYES2 has the 2μ ori which maintains a copy number of 30 to 50 copies per cell. The 2μ ori sequence is derived from the endogenous 2μ circle plasmid (it's the replication origin for 2μ circle).

'CEN' stands for 'CENtromere' sequence. This origin of replication keeps the copy number of that vector down to one or two per cell in yeast. CEN sequences are actual chromosome centromere sequences. CEN ori will keep copy number to 1 or 2 per cell (depending on whether the cell is haploid or diploid). This sequence allows the cell to recognize this plasmid as a chromosome; regulation of chromosome number is extremely rigid. Only functional CEN sequences from S. cerevisiae have been isolated and used on plasmids. CEN sequences are only a couple of hundred base pairs in size. Apparently functional centromere sequences from other eukaryotes, including Sc. pombe and Pichia, are too large to be isolated and utilized on a plasmid.

Answer Id: 3775

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f2b93ce08763fddf54bcb7beb62a2c74_FAQ

What could explain bands of unexpected molecular weight appearing in galactose induced pYES transformants?

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Although there are no other genes on the plasmid that are induced, there certainly are a number of proteins in the cell that are turned on by galactose and any of those may be apparent. As with any experiment, one should always run the plasmid without insert, induced with galactose as a negative control. The additional bands could be due to post-translational modifications, such as glycosylation, that would increase the apparent molecular weight. Since the size of the sugar chains can be variable, glycosylated proteins often appear as less well defined bands compared to non-glycosylated proteins.

Answer Id: 3776

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2119b8d43eafcf353e07d7cb5554170b_FAQ

What is an appropriate innoculum amount to begin a galactose induction experiment? How does raffinose affect the time course of galactose induction?

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The suggested initial cell density for galactose induction is 1 to 5 X 10E6 cells/ml . The cells are allowed to divide one or two times and then induced with galactose. Galactose induction is best in log phase and the culture will probably approach static phase at 1 to 4 X 10E7 cells/ml. Induction of cells maintained in raffinose may begin in 15 to 30 minutes whereas induction of cells maintained in glucose may not first occur for an hour or more. Peak expression will often occur in 2 - 4 hours so time points should be taken every hour (or every other hour) for up to 10 hours. When using raffinose maintained cells, the induction is much faster than induction of glucose maintained cells. Maximal expression levels remain the same.

Answer Id: 3778

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Should D-raffinose be used as carbon source for yeast prior to galactose induction? Does L-raffinose work?

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As with other sugars (e.g. glucose), D-raffinose is the biologically active carbon source for yeast. Pure L-raffinose will not work.

Answer Id: 3779

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8ce87bdda85cd44f14de9afb86491884_FAQ

Is it critical that one uses PEG 4000 for yeast transformations?

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PEG 4000 seems to work best for yeast transformations although PEG 3350 has been used in-house with success.

Answer Id: 3780

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3569df159ec477451530c4455b2a9e86_FAQ

How do I store yeast long term? Can I freeze them at –80°C like bacteria? What is the shelf life of frozen stocks?

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Store yeast frozen at -80°C in 15% glycerol. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws).

Answer Id: 3823

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bca382c81484983f2d437f97d1e141f3_FAQ

How many copies of plasmid per yeast cell are maintained from plasmids with a 2 micron origin of replication?

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The 2 micron plasmid occurs naturally in some strains of S. cerevisae. When a plasmid contains the 2 micron origin of replication it is maintained at 10-40 copies per cell.

Answer Id: 3864

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4f8bc5ac1dc2b49434efe9e72f183de8_FAQ

What is the doubling time of pYES2 transformed S. cerevisiae strain on minimal yeast growth media when either glucose or galactose is used as the carbon source?

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The doubling time of a pYES2 transformed S. cerevisiae strain grown on minimal media with glucose is approximately 2 hours. The doubling time on media with galactose is approximately 4 hours.

Answer Id: 3926

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4fc28b7093b135c21c7183ac07e928a6_FAQ

What are the effects of glucose on the GAL1 promoter in the pYES2 vector?  

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Glucose will shut down completely expression from the GAL1 promoter. Glucose causes repression of the Gal1 promoter.

Answer Id: 3927

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d2dc6368837861b42020ee72b0896182_FAQ

Will S. cerevisiae grow differently using galactose instead of glucose as a carbon source?

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S. cerevisiae can grow using either or both mechanisms of carbon metabolism. The balance between the two is different for glucose vs. galactose as a carbon source. Under ideal conditions, S. cerevisiae grows slower on galactose than on glucose, because production of glucose-6-P from galactose is rate limiting. (gal -> gal-1-P -> glu-1-P -> glu-6-P). Under non-ideal conditions (low oxygen, as in the center of a colony or a culture without really good oxygen feed), it becomes even worse because cells grown on galactose are using more respiration than fermentation relative to cells grown on glucose. Low oxygen makes fermentation more necessary, which cells growing on galactose are not good at.

Answer Id: 4309

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