Product Usage

BL21 (DE3) is different from the latter two. It is a Escherichia coli expression strain used for high-level recombinant protein expression.

DH5α and TOP10 are common Escherichia coli cloning strains. TOP10 (genotype is highly similar to DH10B) retains the mcrA locus, so it may have higher efficiency when transforming methylated DNA from eukaryotic sources (such as cDNA libraries). While the hsdR mutation in DH5α is more suitable for transforming non-methylated DNA obtained through PCR amplification. For the amplification of conventional plasmids, the functions of both can be basically interchangeable.

It is a normal phenomenon that the transformation activity of expression strain competent cells is lower than that of clone strain competent cells, and it is generally within one order of magnitude. Under the premise of proper preservation of competent cells, the number of transformants obtained from normal vectors used in the experiment can meet the needs of most experimental scenarios.

The main difference between the two enzymes is that the E. coli DNA ligase cannot ligate blunt-ended dsDNA fragments. Both enzymes can be used to repair single-strand cuts in double-stranded DNA and perform sticky-end ligation. The E. coli DNA ligase is usually used for cut repair during the synthesis of the second strand cDNA, as the T4 DNA ligase may cause the formation of chimeric insertions.

pPIC9K is an integrated vector that needs to be integrated into the genome of Pichia pastoris through homologous recombination to be stably present and expressed. Depending on the restriction endonuclease used for linearizing the vector, it can be integrated at different sites:

· Using SacI for linearization: Integrate the expression cassette into the AOX1 locus. Obtain the Mut⁺ (normal methanol utilization) phenotype in the GS115 strain; obtain the Mutˢ (slow methanol utilization) phenotype in the KM71 strain.

· Using SalI or StuI for linearization: Integrate the expression cassette into the his4 locus. Obtain the His⁺ Mut⁺ phenotype in the GS115 strain; obtain the His⁺ Mutˢ phenotype in the KM71 strain.

· Using BglII for linearization: Integrate the expression cassette into the AOX1 region of GS115, obtaining the Mutˢ phenotype.

It is not recommended. Restriction enzymes have been formulated with specific concentrations based on the optimal reaction conditions. When diluting the enzyme products, the enzyme concentration and activity do not have a linear relationship. The lower the enzyme concentration, the lower the relative activity, and it becomes more unstable. Sometimes, the substrate DNA may not be cleaved.

The above purification methods can all be used to purify the enzyme-cut products. However, the losses in the phenol/chloroform extraction and gel-cut recovery are relatively large. The intermediate products of stepwise enzyme cleavage can be purified using ethanol precipitation and fragment recovery kits (using within the allowed length range for purification). This operation can reduce the losses.

The λ exonuclease strictly begins digestion from the 5' end of the double-stranded DNA and prefers the phosphorylated 5' end. It has no activity on single-stranded DNA. The T5 exonuclease not only can start digestion from the 5' end of the linear double-stranded DNA, but also can start from the cleavage or nick of circular DNA, and has endonuclease activity for single-stranded DNA, with a wider substrate range.

Both have similar ssDNA binding properties, but ET-SSB exhibits significantly stronger thermal stability due to its presence of a single OB-fold domain and a flexible C-terminal tail structure. E. coli-SSB loses most of its activity above 65℃, while ET-SSB remains fully active after incubation at 95℃ for 60 minutes.

It can be directly added to the reaction system as a PCR additive. It is recommended to mix ET SSB with the template, primers and buffer before the PCR reaction and then perform the thermal cycling. Due to the extremely high thermal stability of ET SSB, it can withstand the high-temperature denaturation step throughout the PCR process without the need for re-addition after each cycle. The specific dosage should be optimized based on the experimental system and a gradient test should be conducted starting from a lower concentration.

For complex templates, ET-SSB stabilizes ssDNA, prevents its formation of secondary structures, and directly or indirectly acts on the polymerase to increase the chain replacement activity and affinity for the primer template, thereby effectively improving the PCR performance of complex templates rich in GC and other types. ET-SSB is also applicable for the optimization of multiplex PCR systems.