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Flashcards in Endonucleases Deck (17):
1

found naturally in

microbes (mainly bactaeria, also archaea)

2

naming endonucleases?

first letter of genus name, first two letters of species name; roman numerals if more than one type of restriction enzymes is made by a particular microbe.

3

whats the recogniition sequence?

specific sequence of nucleotides that the enzyme cuts.

4

recognitions are ...

palindromic

5

why are sticky ends only useful - not blunt ends?

bc they have unpaired bases which can anneal to other unpaired complementary bases.

6

plasmids can be used as

vectors

7

whats a vector?

carrier that transports DNA from an external source (plant/animal) into bacterial cells

8

describe plasmids

CIRCULAR, DOUBle stranded DNA
self replicating (have an origin of replication)

9

how are plasmids formed?

plasmids and foreign DNA cut with the same restruction enzyme.
mixed - joining forming recombinant plasmids (or the same things can join - not reconminant)
ligase enzyme added

10

whats transformation?

when foreign DNA is directly taken up from an external source into bacterial cells.

11

describe transformation of recombinant plasmids into cells

• Electroporation: cells briefly put in an electric field  shocked  pores in membrane  plasmids enter
• Heat shock: plasmids and bacteria placed in salt solution in ice bath  put in 42*C bath for less than a minute  increases fluidity of PM so more plasmids can cross  returned to ice bath  bacteria and antibiotic put on agar plate, ones that haven’t taken up plasmids are killed.

12

describe how copies of the plasmids can be made

• Gene cloning rather than PCR, as its better used when DNA is available in large amounts
• Plasmids replicate  entire bacterial cell replicates via binary fission  copies made.

13

whats gene cloning

locating a gene of interest, and making many copies of it

14

how do we obtain gene of interest

DNA synthesiser
Reverse transcriptase of mRNA remplate

15

describe reverse transccriptase

mRNA template
poly A tail attached
attach primer to poly A
reverse transcriptase forms complementary DNA poylmer
mRNA removed with alkali treatment
normal DNA polymerase...

16

descrube selectable marker genes

-ampicillin resistance gene
-Lac Z - encodes protein that combines with x gel substrate to form blue compound

17

describe how recombinant plasmids are identified and cultured.

added to culture of host bacterial cells (via electroporation/heat shock) which are ampicillin sensitive. ampicillin added. not dead colonies are cultured and proteins are removed and purified.