Protein expression and purification Mammalian cells HeLa

Get tips on using QIAexpress Type IV Kit to perform Protein tag Purification of His-tagged proteins

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Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat mesangial cells

Get tips on using FuGENE® HD Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat mesangial cells

Get tips on using Lipofectamine™ 3000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat schwann cells

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Human prenatal cardiac progenator cells

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. The resulting amplicons are generally detected by gel electrophoresis and for some further applications like cloning, sequencing, amplicon product needs to be recovered from the gel and subsequently purified. However, non-specific product amplification and primer-dimer formation during set-up make gel extraction difficult. Nevertheless, high-quality DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction.

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. The resulting amplicons are generally detected by gel electrophoresis and for some further applications like cloning, sequencing, amplicon product needs to be recovered from the gel and subsequently purified. However, non-specific product amplification and primer-dimer formation during set-up make gel extraction difficult. Nevertheless, high-quality DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction.

Plasmid isolation is an important technique in molecular biology or any kind of genetic editing. It involves amplifying plasmids overnight by transforming them into competent bacterial cells. The desired colonies of these bacteria can then be grown in shaker cultures, at appropriate shaking speed, oxygen availability and temperature. These liquid cultures can then be ultracentrifuged to pellet the bacteria, which are then used for plasmid isolation. The bacteria are first resuspended in a buffer, then lysed, neutralized, purified in a column, eluted, precipitated with ethanol and then resuspended. During plasmid isolation, it is important to lyse cells quickly because lysing bacteria for too long may lead to irreversible denaturing of the plasmid. Usually, alkaline lysis is used for isolation because it is a mild treatment. It isolates plasmid DNA and other cell components such as proteins by breaking cells apart with an alkaline solution. Precipitation removes the proteins, and the plasmid DNA recovers with alcohol precipitation. Resuspension and lysis buffers should be mixed thoroughly in order to prevent the DNA from breaking into smaller fragments. This is because broken gDNA can reanneal and remain in the solution, without binding to the column.