BIOL-4610 Lecture Notes - Lecture 3: Dna Ligase, Crispr, Gel Electrophoresis

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maroonmoose240

25 Apr 2018

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Inserting fragments into vectors - steps: cloning site that has lots of restriction enzyme cut sites, cut plasmid and dna of interest with same restriction enzyme, makes sure that sticky end is exact same. Individually transform each bacterium which holds one of the plasmids: have all the dna represented. In order to make a copy: need a primer, must be specific to gene of interest (~20bp, anneal primer to strands by reducing temperature to 55*c, still hot enough for taq to work. Dna: select using antibiotic resistance from vector, those that ha(cid:448)e(cid:374)"t i(cid:374)tegrated plas(cid:373)id (cid:449)ill die. Introduce an extra gene or transgene: randomly inserted into the chromosome homologous recombination, making a transgenic organism, starting cell is fertilized egg, must be before maternal and paternal genomes combine. February 6, 2018: mutate genes specifically using crispr-cas9, uses a mechanism that evolved naturally in bacteria to protect themselves against phage dna, crispr, clustered regularly interspaced short palindromic repeats, similar to phage dna.

Related Questions

Q1.

You are a genetic engineering hired by the biotechnology company, Genes 'R' Us, to use genetic engineering to develop a new rose that glows. Place the following steps in the order you would need to perform them to successfully genetically engineer the rose with this trait.

-Extract all of the DNA from a firefly cell

-Use traditional plant breeding techniques to improve the performance of the rose, which has the new glowing gene (transgene)

-Clone the single gene that encodes the glowing protein

-Insert the glowing gene into single rose plant cell using either the gene gun or agrobacterium

-Modify the glowing gene so it acts In the way you want it once its inside the rose plants

-Identify the firefly as an organism that would have a gene encoding a protein for a âglowing traitâ

Q2.

Genetic engineering is different from traditional plant breeding in that (select all that apply)

___genetic engineering 'physically' removes DNA (genes) from one organism and inserts them into another

___plant breeding and classical biotechnology relies on natural sexual reproduction to create variation while genetic engineering introduces variation one or a few genes at a time into a single cell.

___genetic variation used in classical biotechnology (plant breeding) comes from mutations in genes already found in that crop species. In genetic engineering, new genes are added to the genetic makeup of a plant

___genetic engineers can move DNA from any organism into any organism, even if they are not in the same species.

Q3.

Which method of plant transformation works and depends upon an organism that is a "naturally occurring" genetic engineer?

A)Gene Gun

B)Agrobacterium

C)Microneedle insertion into a plant egg cell

D)Electric Shock of Plant Cells

rubydeer239

1. A study was conduced to identify a liver cell gene (zed) that has a length of 3000 bp. A southern blot was prepared using DNA from the liver cell and a probe made to region 1600-2000 of zed. Analysis of the blot revealed that there are two spots present, one at 3000 and another at 2000. A plausible explanation for this observation is

A. there is an identical copy of this gene on another chromosome.

B. the gene was cut into two equal portions by a restriction enzyme.

C. there is another gene with sequence similar to gene zed

D. both: there is an identical copy of this gene on another chromosome and there is another gene with sequence similar to gene zed

E. no explanation can be given using the available data

2. Protein P is synthesized in relatively high amounts in human pancreas. This protein has been isolated and purified, but its amino acid sequence has not been determined. We wish to clone the gene for protein P.

(a) How can a probe be prepared to identify the gene for protein P?

(b) If we have prepared a radioactive messenger RNA as our probe in part(a), how could we verify that it is the mRNA for protein P?

(c) If we wish to make a gene library from the DNA of human pancreatic cells, we proceed by digesting the DNA with the restriction enzyme BamHI and then separating the fragments by agarose gel electrophoresis. The fragments are transfered (by southern blotting) onto a nitrocellulose filter and flooded with the radioactive probe. Following incubation, the filter is washed to remove any probe that has not specifically bound to one or more DNA fragments. Autoradiography of the filter reveals two bands of size 300 and 700 bp. When experiment is repeated using restraction enzyme PstI, only one band of 1000 bp appears. If it is already known that pancreatic cells contain a single copy of the gene of protein P, which of these enzymes should be used to construct our gene library?

violethornet799

I need to make an antibody for the extracellular portion of the Notch receptor. I have made S35 labelled Delta and my boss has given me an antibody that recognizes Delta and is conjugated to a chromophore that gives off light. Place the following statements in order beginning with labeling. HINT: Labeling, protein enrichment, immune response, making monoclonal cell lines, antigen screening of monoclonal cell lines.

A) Hybridoma cells are selected by plating them in HAT buffer.

B) Using direct ELISA you coat individual tissue culture wells/dishes with a single monoclonal antibody.

C) I detect my protein fraction using a geiger counter.

D) You add a solution containing radiolabeled Notch extracellular domain to the wells.

E) You only keep monoclonal hybridoma cell lines that give light. The other cell lines you throw away. You have your antibody!

F) I centrifuge my homogenate in a sugar or salt gradient.

G) I inject a mouse with my protein, giving booster shoots every week.

H) Heterokaryon cells are generated by the fusion of plasma cells and a myeloma cell line.

I) You wash away excess Delta antibody.

J) You add medium containing the Delta antibody that is covalently bound to a chromophore to the individual wells of the ELIZA.

K) To determine if the monoclonal antibody from any given monoclonal cell line recognizes the Notch extracellular domain, you look for light.

L) I use dialysis to remove excess salt and or sugar and to place the protein into a solution that mimics that of the cytoplasm.

M) The mouse is euthanized to harvest the plasma cells in the spleen.

N) Vast quantities of the exported form of the triggered antibody from plasma cells are released.

O) On some B cells, individual antibody receptor forms are bound by the Notch antigen for and activate B cells.

P) I fractionate the proteins.

Q) Individual hybridoma cells are cultured in separate tissue culture wells/dishes to create monoclonal cell lines. These lines export the unique antibody they make into the culture medium.

R) I take culture cells with the Notch receptor expressed on the cell surface and mix them with radio-labelled Delta.

S) B cells divide to give rise to plasma cells and memory cells.

T) You wash away excess Notch extracellular domain from the coated ELISA wells

BIOL-4610 Lecture Notes - Lecture 3: Dna Ligase, Crispr, Gel Electrophoresis

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