Microcon ym-10 manual

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Millipore microcon centrifugal filter devices Microcon centrifugal filter devices as described in Materials and Methods. Millipore ym microcon centrifugal filter Microcon centrifugal filter devices as described in Materials and Methods.

The mixture was subjected to centrifugal ultrafiltration using Microcon YM Millipore to obtain Article Snippet: The reaction mixture was loaded onto Microcon YM Millipore and centrifuged for 10 min at 10 g.

Techniques: In Vitro, Incubation. Inocula were transferred Article Snippet: Biofilms were cultured in Microcon centrifugal filter devices catalog no. Using enhanced green fluorescence protein as the model system and deoxyinosine as the universal base, we proved by sequencing genes the concept of the SeSaM method and achieved a random distribution of mutations with the mutational bias expected for deoxyinosine. Inspired by Darwinian evolution in nature, directed evolution methods have been developed for tailoring proteins to our needs and elucidating structure—function relationships 1.

An important step in a directed evolution experiment is to efficiently explore sequence space through random mutagenesis. The sequence space of a truly randomized library is, by its nature, not limited by a preselection for function under physiological conditions and allows us to adapt proteins to non-natural environments such as biotechnological processes.

Among random mutagenesis methods, error-prone PCR epPCR methods based on inaccurate amplification of genes have been very successful and are generally employed in directed enzyme evolution experiments due to their simplicity and versatility 1. Though versatile and simple, all known epPCR methods are significantly limited in their ability to create diversity on the gene level 9.

Instead of changing one amino acid to all other 19 possible amino acids, an average of less than five other amino acids can be obtained with current epPCR methods. The reasons lie mainly in the redundant genetic code and the biased mutational spectra of DNA polymerases 7.

Additionally, most directed evolution experiments are performed at low to moderate mutation frequencies in order to avoid a large portion of inactive clones. Thus only 1—3 nt have been mutated per bp 7. Statistically these mutations rarely occur next to each other. Furthermore, mutational bias of the employed DNA polymerases [methods a , c and d ] significantly reduces the diversity of the nucleotide substitutions and their random incorporation in the target gene.

For instance The incorporation of nucleotide analogs occurs in a PCR at preferential sites and results in reduced diversity [methods c and d 4 , 5 ]. An additional drawback of these methods is the inefficient incorporation of nucleotide analogs by polymerases 10 , resulting in limited mutation frequencies 11 and often low product yields.

As a truly random mutagenesis method, sequence saturation mutagenesis SeSaM overcomes limitations caused by biased polymerases. Moreover, SeSaM can target a nucleotide species of the selected sequence and each nucleotide species can be exchanged in a controlled manner since SeSaM regulates the mutational spectra through a universal base.

The single-stranded template was released from Dynabeads according to the manual by boiling in 0. The sample reservoir was inverted and spun at 14 g for 3 min to recover the desalted DNA.

The mixture was incubated at room temperature for 1 h. Desalination was done using the NucleoTrap kit Macherey-Nagel. The mixture was incubated at room temperature for 10 min with gentle shaking every 2—3 min. The suspension was centrifuged with NucleoSpin Microfilter Machery-Nagel at 10 g for 30 s to separate silica matrix from the DNA-containing solution.

Each reaction mixture consisted of: 5 U terminal transferase New England Biolabs , 0. The cells were plated and grown on LB amp plates. SeSaM is a four-step method that can saturate every single nucleotide position of the target sequence with all four standard nucleotides Fig.

In the first step a pool of DNA fragments with a random length distribution is generated. In the third step the elongated DNA fragments are extended in PCR to the full-length genes by using a single-stranded template and a reverse primer. In the fourth step a concluding PCR is used to replace the universal bases with standard nucleotides. Scheme of the SeSaM method that comprises four steps.

Step 1: creating a pool of DNA fragments with a random size distribution. Step 2: enzymic elongation of the DNA fragments with a universal base. Step 3: full-length gene synthesis. Step 4: universal base replacement by standard nucleotides. In the first step Fig. Separation of this library from cleaved products is achieved by isolating biotinylated forward primer using magnetic particles with a streptavidin-coated surface in a DNA melting solution.

For the success of this method, it is important to fully remove nicked complementary strands in this purification step. Subsequent release of the biotinylated DNA fragments from the solid support is achieved by boiling in 0. A scheme of Step 1. B Gel pictures: PCR products before left lane and after right lane iodine cleavage. In the second step Fig. Deoxyinosine is a well-studied universal base with a strong preference for base pairing with cytosine 11 , It is used as a replacement for guanine in DNA sequencing 17 ,