Protein can be directly quantitated by colorimetric. Coomassie Blue dye binds to peptides in an acidic medium. The color change of this assay is measured at 595 or 600 nm. We calculate the protein concentration using the formula created by standards.
Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity while using very simple and cheap equipment and chemicals. The procedures of silver staining are protein fixation, silver impregnation and image development.
Bradford assay is a rapid and accurate method to determine the concentration of protein. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. Within the linear range of the assay (~5-25 mg/ml), the more protein present, the more Coomassie binds.
Following PCR, you need to get rid of excess short primers, dNTPs, enzymes, short-failed PCR products and salts. We use a silica-gel-membrane for binding of DNA in high-salt buffer (pH 4.5~5.5) and elution of DNA with low-salt buffer (pH 7.0~9.0) or ddH2O to get clean PCR products for downstream applications.
Digestion is the process of cutting DNA molecules with restriction endonucleases. These special enzymes recognize specific sequences in the DNA molecule (for example GATATC) wherever that sequence occurs in the DNA. In PCR cloning, insert and vector are cutted by the same restriction endonucleases and then join together in the next step, ligation.
The purpose of transformation is to introduce a foreign plasmid into bacteria and the bacteria will replicate the foreign plasmid along with their own DNA. Bacteria which are able to uptake DNA after a heat shock are called "competent".
It can be used after a transformation to screen colonies for the plasmid. Primers designed for the insert sequence should be used when preparing the PCR reaction. Thus, any colonies which give rise to an amplification product are likely to contain the correct DNA sequence.
PCR cloning is a method of cloning which dramatically reduces the time and effort put into the cloning reaction. PCR cloning procedure consisting of the four following steps: (1) production of a fragment of the gene using PCR, (2) digestion of genomic DNA, (3) ligation into a plasmid vector, and (4) transformation into bacteria and then bacteria will replicate the plasmid.
Polymerase Chain Reaction (PCR) is a technique to amplify few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. There are three major steps (Denaturation, Annealing and Extension) in a PCR and repeat for 30 or 40 cycles.
Automated protein synthesizer is an expert system in transcription, translation, and purification of recombinant protein. It is based on the eukaryotic translation apparatus of wheat germ. This high throughput system produces recombinant proteins in vitro. The generated proteins display foldings and biological functions similar to the mammalian counterparts.
A plasmid DNA extraction is a method used to extract and purify plasmid DNA. Kits are available from varying manufacturers, which are named by size of bacterial culture. Miniprep is used when the starting E. coli culture volume is 1~5 ml of LB broth and the expected DNA yield is 20~30 μg.
Protein can be directly quantitated using UV absorbance (280nm). 280nm Absorbance depends on the Tyr and Trp (amino acids with aromatic rings) content. A very rough protein concentration can be obtained by making the assumption that the protein sample has an extinction coefficient of 1 (1 OD = 1 mg/ml protein).
Alkaline lysis is one of the most generally useful methods for isolating circular plasmid DNA from bacterial cells. This video demonstrates the procedures for alkaline lysis method.
PCR can be used to make changes to the nucleotide sequence of DNA. This is called PCR mutagenesis. Site-direct mutagenesis is one kind of PCR mutagenesis, which introduces a mutation at a specific location on the DNA strand.
Protemist® XE is a fully-automated desktop protein synthesizer for large scale protein production using the wheat germ cell-free expression system. It is ideally suited for producing 10 mg to over 500 mg of protein for structural analysis, functional analysis, and assay development.
Dr. Tomohisa Satoh presented two new generation, fully automated robotic desktop wheat germ cell-free protein synthesizers : Protemist® DT II (High Throughput) and Protemist® XE (Large-scale).
The demonstration for Protemist® DT II - High Throughput Protein Expression and Protemist® XE - Large-Scale Protein Expression.
A plasmid DNA extraction is a method used to extract and purify plasmid DNA. Kits are available from varying manufacturers, which are named by size of bacterial culture. Midiprep is used when the starting E. coli culture volume is 15-25 ml of LB broth and the expected DNA yield is 100-350 μg.
A plasmid DNA extraction is a method used to extract and purify plasmid DNA. Kits are available from varying manufacturers, which are named by size of bacterial culture. Gigaprep is used when the starting E. coli culture volume is 2.5~5 L of LB broth and the expected DNA yield is 7.5~10 mg.
Human embryonic stem cells (hESCs) are pluripotent cells are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. This video describes how to prepare cryopreserved hESCs for long-term storage.