SCIENCE DIARY

mini prep of YEp552 vector

260/280: ratio of sample absorbance at 260 and 280 nm. The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. See “260/280 Ratio” section of the Troubleshooting section for more details on factors that can affect this ratio. 

For obtain human liver cell cDNA library in YEp552 vector from E.coli

User guide of  “ PureLink® HiPure Plasmid Filter Purification Kits For Midi and Maxi preparation of Plasmid DNA “ written below:

- The HiPure technology, based on anion-exchange chromatography, uses a patented resin composed of small particles with a uniform pore size.

-Column equilibrated with EQ1. E. coli cells are harvested with a centrifuge, then resuspended in Resuspension Buffer (R3) with RNase A, and lysed with Lysis Buffer (L7, SDS/alkaline lysis). Precipitation Buffer (N3 P.Ac) is then added to the lysate. The lysate is poured into a pre-packed anion-exchange column fitted with the Filtration Cartridge unit. In one simple combined step, the lysate is clarified, and the negatively charged phosphates of the DNA backbone interact with the positive charges on the surface of the anionexchange resin. The temperature, salt concentration, and pH of the solutions are optimized for efficient binding of DNA. A single column wash under moderate salt conditions using Wash Buffer (W8) removes RNA, proteins, carbohydrates and other impurities while the plasmid DNA remains bound to the resin. The plasmid DNA is eluted under high salt conditions with the Elution Buffer (Tris-HCl +NaCl). The eluted DNA is desalted and concentrated by alcohol precipitation. TE buffer used to resuspend the DNA pellet.

Isopropanol Precipitation: The salts neutralize the negative charge of the negatively charged phosphate in DNA and the isopropanol /ethanol removes the hydration shell of H2O molecules around the phosphate.

I will perform transformation with a mix. This is the recipe: 

PEG: is essential for attachment of DNA to the surface of the cell. Also covers cell membrane and protects it from chemical lysis. 

Lithium Acetate: Li ions permabilizes cell wall.

EDTA: to inhibit DNase activity. 

Tris: to buffer the solution

dH2O

DTT: as reducing agent that used to reduce disulfide bridges within proteins. It is believed that it distrupt membrane proteins (ion channels, receptor proteins etc.) and modify cell surfaces. That allows more cells to take up DNA. (may be lethal for cells) 

Spectrophotometer is a commonly used technique to measure absorbance of DNA or nucleics acids relative to wavelenght. 0.5 – 2 µL of sample is enough and it only took 10 seconds to quantify.  For nucleic acid quantification, the Beer-Lambert equation is modified to use a factor with units of ng-cm/microliter.

Here, we used it to determine vector (plasmid) and human liver cell cDNA library concentration.

cDNA library

YEp352 vector. 

The spectral display shows data for the current sample normalized to a 10 mm path for all measurements including measurements made with any cuvette pathlength. 

The following features are to the right of the spectral display: 

 • Sample ID - field into which a sample ID is entered. The appropriate sample ID should be entered prior to each measurement. 

 • Conc - concentration based on absorbance at 260 nm and the default or user defined extinction coefficient. 

• A260 - displays absorbance at 260 nm normalized to a 10 mm pathlength. 

• A280 - displays absorbance at 280 nm normalized to a 10 mm pathlength. 

• 260/280 - ratio of absorbance at 260 nm and 280 nm. The ratio of absorbance at 260 and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. 

This graphic also allows us to determinate purity of the sample, nucleic acids have the maximum absorbance at 260-280 nm, there is not another peaks at other wavelenghts, so there isn’t any protein contamination.