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Flashcards in Molecular Techniques Deck (35):
1

How to Clone

- isolated wild type genomic DNA
- cut up DNA with restriction endonuclease
- clone DNA into a plasmid

2

DNA cut up by endonuclease

- foreign DNA (unmethylated) is cut up by restriction modification system - endonuclease and methylase
- will target the hemimethylated or unmethylated DNA because all DNA in the cell will be methylated.

3

resistance markers

- grow in media with antibitoic
- only cells with antibiotic resistance plasmid will grow

4

addiction modules

- cells grown in poison
- plasmid contain antidote gene
- poison is long lasting
- antidote is short lived
- must replicate plasmid to live

5

EcoRI type II

- methylase - methyl group added to the 2nd adenine residue
- if methylated, EcoRI endonuclease will not cut it
- unmethylated sequence will be quickly cut up by EcoRI endonuclease.

6

Classes of restriction endonucleases

- Class I
- Class II
- Class III

7

Class I

- multisubunit enzyme that requires SAM, ATP, and Mg2+
- cut DNA at distant site

8

Class II

- only require Mg2+ and usually cut with palindromic recognition site

9

Class III

- requires ATP
- not commercially available

10

Class I mechanism

- enzyme binds to recognition site
- DNA translocated through enzyme and cut at the distant site
- methylate and nuclease are part of the same complex (why they require SAM)

11

Using Restriction enzymes in cloning

- over 300 available
- each one cuts at a unique DNA sequence
- most 4 to 6 base pair palindromic sequences

12

Steps to cloning with restriction enzymes

1. extract DNA from bacteria
2. cut DNA with restriction enzyme
3. cut vector with same restriction enzyme
4. incubate DNA fragments with vector and seal with ligase + ATP to seal phosphate backbone
5. transform ligated products into E. Coli
6. grow bacteria on plates containing antibiotic
(whatever resistance cloned for)
7. screen the colonies for inserted DNA

13

When DNA is cut by restriction enzyme

- it is staggered
- only staggered ends cut with same restriction enzyme will go back together

14

plasmid issue

- each colony that grows will carry a plasmid
- the problem is the plasmid has compatible ends with itself
- selection for those with correct insert.

15

Blue White/Screening

-Uses the lacZ gene product (B-galactosidase) to determine if a piece of DNA has been cloned into the multiple cloning site of a vector.
- the multiple cloning site to cut plasmid to insert DNA is in middle of LacZ gene
- plasmids that have this insert do not make an inactive B-galactosidase enzyme
- plasmids that do not can still produce the enzyme
- put X-gal on plates, cells with empty plasmid will be blue.
- colonies that have plasmids containing the inserted will be white

16

How to fish out interesting genes

- sequence all inserts
- DNA hybridization if you know the sequence
- screen for insert activity
- if you know insert activity, insert that into mutant and screen for it.

17

Typical Assays

- enzyme activity
- activity of a "reporter"
- mRNA concentration
- protein concentration (western Blot)

18

reporter gene

coding region for the gene

19

promoter activity

- reporter fusions
- if studying the regulation of a protein that is not easy to assay, replace it with a protein that is easy to assay.

20

Reporter gene protocol

1. clone promotor for gene you are studying
2. clone the coding region for the reporter gene downstream of promotor
3. put your reporter construct into organism
4. grow cultures under the conditions the should should be on or off
5. assay reporter gene.

21

B-galactosidase as a reporter gene

- use calorimetric assay

22

calorimetric assay

- the products of the assay produce a color
- use Xgal to determine if active in plates (blue/white)
- use ONPG which turns yellow when cleaved by B-galactosidase
- quantify using a spectrophotometer.

23

Regulation with lacZ

- w/o trp - activation of trpEDCBA genes to make Trp
- w/ trp - repression of trpEDCBA genes
- TrpE is an anthranilate synthase (hard to measure
- replace with LacZ
- grow cells +/- tryptophan
- Add ITPG or X-gal
- measure amount of product with a spectrophotometer.

24

GFP

- isolted in the jellyfish Aequora victoria which glows green
- small and folds easily so it is active in most bacteria
- fuse the coding region to the promotor you are studying and then look for fluorescence.
- since the substrate is light, you can view in live cells.

25

methods of direct mRNA measurement

- northern blot
- qrtPCR

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northern blot

- RNA subjected to electrophoresis through agarose gel
- separate all polymers based on size
- label a DNA sequence, and use it to probe an RNA blot
- RNA/DNA hybrid forms
- band appears darker when more mRNA is present.
- does not tell you absolute quantity

27

qRT-PCR

- make DNA copy of RNA
- bind specific DNA oligonucleotide primer complementary to 3' end of mRNA and extend with RTase
- after DNA made add a second primer to further DNA amplificaiton
- takes place in presence of sybr-GRN
- fluoresces when bound to dsDNA

28

intensity threshold

-assigned
- above background but before log phase

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cycle #

inversely proportional to amount of starting mRNA

30

quantification

- takes place in thermocycler that monitor flour
- computer graphs fluoro versus cycle number

31

Western Blot

- measures amount of protein expressed
- uses an antibody against the protein

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advantage of western blot

sometimes the amount of protein expressed is not entirely dependent on the mRNA expressed

33

SDS-PAGE

- separates cellular proteins
- each protein denatured and given a negative charge with detergent
- given a uniform negative charge/mass ratio
- loaded on top of gel and electrical field applied
- all proteins migrate toward + electrode, the smaller proteins move fastest

34

primary antibody

antibody that recognizes your protein

35

secondary antibody

- the antibody that is used in the blotting
- usually radioactive
- visualize with this one
- directed against antibody constant region - same for all antibodies in a species.