Weeks 3/4 Notes

Seite 1

add a chemical to induce

Induction by IPTG is like induction with the lac operon

There is a repressor in the way that prevents RNA polymerase from reading the rest of the strand. Lactose binds to the repressor and causes it to detatch from the operator.

Ligation: process of joining two DNA strands or other molecules by phosphate ester linkage

Restriction Enzymes: selectively cut DNA withing the recgonitions site by cleaving a phosphodiester bond within the DNA backbone

Blunt ends have no overlap

Sticky ends have overlap

To construct Abl containing vector... cut both desired DNA fragmetn and Vector with the same restriction enzymes and incubate together. Use two so the DNA doesn't insert backwards

then used DNA ligase to connect 5' and 3' ends to create a continuous plasmid

We used Xba1 and Xho1 to cut

How to check that you were sent the correct vector:

DNA sequencing (confirms sequence and is most thorough)

Restriction digestion (confirms insert size and location

in a gel, less intense bands may just mean smaller

UV spec... aromatic rings primarily responsible for UV light absorbance

DNA has a higher extinction coefficient that proteins

it takes a ton of proteins to contaminate a DNA substance enough to significantly change the absorabcne of the DNA sample at