Gel electrophoresis

Electrode travels to positive in gel electrophoresis, smaller molecules travel faster because it is easier (gel has pores so smaller molecules travel farther since it is easier for them to navigate)

Recombinant DNA- 

combining DNA from different sources

1) cut them with restriction enzymes with the type that gives sticky/single-stranded ends

use the same restriction enzyme so we get the same complimentary sticky ends

cut the human DNA with the restriction enzyme, and cut plasmid DNA with the same restriction enzyme

sometimes the plasmid closes up on itself and it might not get picked up

ligase connects sugar phosphate diester bonds

properties of plasmids-

separate from the bacterial chromosome

have their own origin or replication

circular pieces of DNA

small amounts of DNA

plasmids are one of the main vectors we use in order to accept a piece of DNA (create recombinant DNA)

Transformation process-

transforming or changing the phenotype of bacteria by incorporating foreign DNA

(makes it a more efficient process by making bacteria more likely to pick up plasmids: competent)

-heat shock and calcium chloride (ice heat ice) opens up pores in the cell walls: facilitating plasmid to go in

Selection-

two types of selection:

Blue-white selection

vs

antibiotic selection

vectors

-recognize that when yeast is used, it is better for expressing human genes (protein folding?)

-the relative order of vectors

cDNA library

c stands for complimentary

The human genomic library cuts up DNA into small pieces, inserts into some kind of vector that then goes into bacteria to be stored (any part of the DNA of a genome of a particular organism)

(cut up DNA and insert into plasmids)

cDNA library is more about picking a specific gene i wanna put into a library and more importantly the DNA is made from messenger RNA (has a poly-A tail which tells that it has been to RNA processing, so the introns have been removed so it can go right to gene expression if we wanted to do that

(more specific)

1) DNA copy of RNA is made

2) second copy of DNA

DNA probes:

A sequence that is complimentary to some kind of sequence we’re looking for in the library

Mainly what happens- if DNA is in a library and you’re looking for a specific gene, we can heat the sample to make it denature, if we know the gene we are looking for we can add it into the sample, and the probes have different tags that have either an enzyme that gives some kind of reaction (fluorescent, some kind of signal), if the sequence we are looking for is present in the library sample it will bind to it and it will give some kind of indicator that what were looking for is there

-they’re complementary to whatever it is were looking for

-has some kinda tag that tells that it has bound, meaning the sequence is present

if we have a library where a bunch of DNA in plasmids is there

If there is a specific gene we want to find we can use a gene probe to do so

PCR:

functions of all individual components

DNA polymerase- adds the nucleotides to extend our strands

primers- start it off, initiate a strand, gives something to add on to

source of four nucleotides?

why is taq polymerase important: heat resistant so that when we do the denaturation step and we heat so separate the strands, it itself is not denatured and so it can build the new strands

-can be used for genetic testing, forensic analysis, used in any situation where we have little DNA and want to make a lot of DNA