Invitrogen™ CSM Media for Mav203 Yeast Cells

Catalog No.	Quantity
A13292	2 L

Catalog No. A13292 Supplier Invitrogen™ Supplier No. A13292

Description

A specifically designed media for making agar plates that enable the growth of Saccharomyces cerevisae MaV203 cells

The CSM Media for Mav203 Yeast Cells is a specifically designed media for making agar plates that enable the growth of Saccharomyces cerevisae MaV203 cells. This product comes with enough reagents to prepare 2 liters of solid media with the following components per liter:

• CSM (without tryptophan): 0.74g
• Yeast Nitrogen Base: 6.66g
• Ammonium Sulfate: 5g
• Glucose: 20g
• Agar: 20g

Compatible with All MaV203 Applications
The CSM Media is optimized for the GeneArt™ High-Order Genetic Assembly System, including the recovery for transformations of vectors once they have been adapted to yeast using the GeneArt™ High-Order Vector Conversion Cassette. The media can also be used to grow MaV203 competent yeast cells, both library and sub-cloning scale (cat# 11281-011 and 11445-012).

Plate Preparation Protocol
For each liter, use one pouch of CSM -Trp Agar Yeast Media minus Glucose and 1 bottle of 20% Glucose. Dissolve the contents of the pouch in 900mL of distilled water and autoclave for 20 minutes on liquid cycle. Allow the solution to cool to 55°C and then add one bottle of 20% Glucose (100mL). Mix well and pour plates.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

• Content And Storage: CSM Media for MaV203 Yeast Cells has enough reagents to prepare 2 liters of agar media. The product contains two pouches of premixed powder and two 100 ml bottles of filter-sterilized, 20% glucose.
  
  Store all components at room temperature.
  
  All components are guaranteed stable for 6 months when properly stored.
• Form: Liquid, Powder
• Media Type: CSM Media
• Preparation Method: Autoclave
• Product Type: Microbiology Media
• Sterility: Non-sterile
• Target Organism Class: S. cerevisiae
• Final Product Type: Agar Plates
• For Use With (Application): Optimized for use with the GENEART™ High-Order Genetic Assembly System, A13285
• Product Line: GeneArt
• Quantity: 2 L
• Shipping Condition: Room Temperature
• Strain Designations: MaV203

Frequently Asked Questions (FAQs)

Can I purchase the CSM Media from the GeneArt High-Order Genetic Assembly System separately?

The CSM Media is available as a standalone item (Cat. No. A13292).

With the GeneArt High-Order Genetic Assembly System, I'm getting no positive colonies detected by yeast colony PCR. Can you please offer some troubleshooting tips?

We would recommend trying to re-streak the colony on a fresh plate and repeat colony PCR. Do not break open the yeast cells with the beads supplied with the kit; the beads are for transformation into E. coli. Additionally, use less than 0.5 µL of diluted yeast lysate in a 50 µL PCR reaction.

With the GeneArt High-Order Genetic Assembly System, I see small or no yeast colonies after transformation. Can you please offer some suggestions?

Ensure that yeast transformations are incubated at 30 degrees C for 3 days for proper colony formation.

With the GeneArt High-Order Genetic Assembly System, I did not get any yeast colonies after transformation and the transformation control did not work. Can you please offer some suggestions?

Please review the following suggestions:
– Perform transformation exactly as described in protocol.
– Do not freeze-thaw or vortex MaV203 yeast competent cells.
– Use CSM-Trp agar plates for the transformation.
– For best results, use fresh DMSO from an unopened bottle. You may use DMSO stored at -20 degrees C.

I want to use my own E. coli vector for my assembly. Can I do this? How?

Yes, you should be able to adapt your E. coli vector into a yeast-compatible cloning vector using the GeneArt High-Order Vector Conversion Cassette (Cat. No. A13291) for use with the GeneArt High-Order Genetic Assembly System with the following provisions:
– Start by using the DNA Oligo Designer web tool, and verify that your vector and the GeneArt High-Order Vector Conversion Cassette do not share internal homology to prevent potential re-arrangements when using your adapted vector with the GeneArt High-Order Genetic Assembly System.
– Use a vector with a single- or low-copy-number origin for a final construct of >15 kb, if the final plasmid construct will be transferred into E. coli. Usually, low-copy-number E. coli vectors have significantly higher capacity than high-copy number vectors.
– Avoid chloramphenicol selection markers on the custom vector since this is the marker on the cassette.
– After ligation (1:10 vector: insert ratio recommended), transform competent E. coli cells with the ligation mixture and plate on double selection LB plates (chloramphenicol plus the antibiotic marker on your custom vector backbone).To linearize your yeast-adapted cloning vector for multi-fragment assembly, a double-digestion is required to avoid background caused by residual palindromic end sequences resulting from a single enzyme digestion.

With the GeneArt High-Order Genetic Assembly System, what are the requirements for stitching oligonucleotides used for insertion editing versus deletion editing?

Stitching oligonucleotides used for insertion editing must have a 30-nucleotide overlap with each adjacent fragment in addition to the insertion bases (for a total length of up to 80-mer, including up to 20 insertion bases). See manual for diagram. Note: This applies for a 2-fragment assembly and the insertion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.


Stitching oligonucleotides used for deletion editing must have a 40-nucleotide overlap with each adjacent fragment, annealing up to 6 nucleotides from the junction into each fragment, thus leaving up to 6 bp at the end of each fragment to be deleted during transformation-associated recombination. See manual for diagram. Note: This applies for a 2-fragment assembly and the deletion applies only to the internal junction. Use a 40-bp overlap (i.e., an 80-mer oligonucleotide) for the remaining seamless junctions.

For the GeneArt High-Order Genetic Assembly System, what enzyme do you recommend for amplifying my DNA fragments?

We recommend using our Platinum PCR Supermix High Fidelity enzyme (Cat. No. 12532-016) for amplifying DNA fragments up to 5 kb for general assembly applications. If you require higher PCR fidelity, we would recommend one that has processing and proof-reading capabilities.

I am using the GeneArt High-Order Genetic Assembly System and am trying to assemble large pieces of DNA. Do you have any recommendations?

Large fragments (>5 kb) are more susceptible to damage in a gel extraction procedure. Therefore, when using DNA fragments generated by PCR, we recommend that you assemble multiple fragments of less than 5 kb in one reaction rather than a single large fragment. Additionally, try to minimize UV exposure of the DNA and/or limit EtBr amount used. If you are attaching the insert to the linearized cloning vector, all nucleotides providing the requisite homology must be on the 5' end of the primer. If you are connecting two adjacent inserts, you may split the 30- to 50-bp homology between the fragments (e.g., 15 bp on the reverse primer of fragment 1 and 15 bp on the forward primer of fragment 2 for a 30-bp homology, or 25 bp on the reverse primer of fragment 1 and 25 bp on the forward primer of fragment 2 for a 50-bp homology). Please note, you can split the homology between adjacent fragments in any combination (e.g., 15 + 15 as in the example above or 20 + 10, 25 + 5 etc. for a 30-bp homology).

What are the specifications for the pYES1L vector? Can I use this as my cloning vector?

The pYES1L vector, a 9.4 kb linearized YAC-BAC shuttle plasmid, is a control vector for the assembly of DNA fragments in MaV203 cells and for the transfer of the assembled recombinant DNA molecule into E. coli. You can also use the pYES1L vector as a cloning vector, if desired. The origin of replication for yeast is chromosome II's centromere (single copy, high capacity YAC). The origin of replication for E. coli is F' ori (single copy, high capacity BAC). Spectinomycin is used for selection in E. coli. Four reactions (1 tube with 8µL) of the pYES1L vector are included with the kit. This is enough for control reactions, for cloning your inserts, or a combination of both. The pYES1L vector is also available separately (Cat. No. A13287, 10 reactions).

Will the addition of A-overhangs by the PCR enzyme affect cloning efficiency with the GeneArt High-Order Genetic Assembly System?

No, you can use enzymes that leave blunt ends or A-overhangs. Cloning efficiency will not be affected.

What cloning efficiency can be expected using the GeneArt High-Order Genetic Assembly System?

Cloning efficiencies can vary greatly based on the number of fragments with end-homology. Below are the approximate cloning efficiencies of assembled fragments into pYES1L:
>90% for 5 DNA fragments of 10 kb each
>90% for 10 DNA fragments of 5 kb each
>50% for 10 DNA fragments of 10 kb each
For pre-existing fragments without end-homology assembled into pYES1L using ‘stitching' DNA oligonucleotides, common cloning efficiencies are:
>90% for 1 fragment of 10 kb
>75% for 2 DNA fragments of 10 kb each

How do I use the web-based tool for fragment editing with the GeneArt High-Order Genetic Assembly System?

The web tool is not set up to do fragment editing automatically, but it can be used to do it manually. The manual has a section on fragment editing that gives examples on how to use the tool for this purpose.

I'm cloning 4 DNA fragments in a vector. Should I use the GeneArt Seamless Cloning and Assembly (Plus) Kit or the GeneArt High-Order Genetic Assembly Kit?

If the fragments are all below 5 kb and the total size of the molecule is below 13 kb, we would recommend the GeneArt Seamless Cloning and Assembly (Plus) kit. If you are assembling elements that have no end-homology, are too large to be amplified by PCR, or are trying to create a molecule over 13 kb, we recommend the GeneArt High-Order Genetic Assembly System. Overall, the High-Order system can do more fragments, fragment editing, and oligonucleotide stitching and can do it at a higher efficiency. However, the assembly is in vivo (yeast) and takes longer to get the final construct due to longer growth periods for yeast compared to E. coli. Select the GeneArt Seamless Cloning and Assembly kit that is best for your application by visiting http://www.thermofisher.com/us/en/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html.

With the GeneArt High-Order Genetic Assembly System, can I assemble a molecule with fragments that carry homology (PCR) and others that are pre-existing (stitching) at the same time?

Yes, the kit can assemble up to 5 fragments and 3 oligo stitches.

Do you recommend a particular purity of oligonucleotides for the GeneArt High-Order Genetic Assembly System?

We have developed the kit using desalted oligonucleotides, but higher purities may result in slightly better cloning efficiencies, especially during fragment editing.

With the GeneArt High-Order Genetic Assembly System, how many overlapping nucleotides must I have if my construct is larger than 60 kb?

We recommend having a total overlap of 50 nucleotides with adjacent fragments for constructs larger than 60 kb. For constructs smaller than 60 kb, a total overlap of 30 nucleotides is recommended.

Why does my prestained standard give different molecular weights for different types of gels?

Protein gels vary in pH. Each of the proteins in the standard has a dye molecule attached to it, and the charge on these dye molecules can change depending on the pH of the gel. This change in charge will affect the migration of the protein on the gel.

For Research Use Only. Not for use in diagnostic procedures.