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Flashcards in Enzymes and Primers Deck (21)
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1
Q

Why is CIP used in plasmid cloning?

A

Removes 5’ and 3’ phosphate of linear DNA to prevent religation with speed and high specificity.

2
Q

Problems with CIP

A
  1. Difficult to eliminate via heat denaturation
  2. Sticks to DNA ends
  3. If residual CIP carries through to ligation, insert will be dephosphorylated
  4. Too much will damage DNA
3
Q

Why is SAP a good alternative to CIP?

A

Rapid irreversible heat inactivation at 65C

4
Q

Polymerase I was orinigally purified in _____ but ubiquitous in ______ year _____

A

E.coli, prokaryotes, 1956

5
Q

How is the klenow fragment made?

A

Domain necessary to code for 5’-3’ exonuclease activity removed by proteolytic treatment of PolI

6
Q

Problems with using klenow

A

New enzyme would have to be added every cycle

7
Q

Taq can be used for ___ min at ___ or ____ at ___

A

30 , 95, 9, 97.5

8
Q

Problem with Taq

A

no 3’-5’ exonuclease activity

9
Q

What does Pol I need for dNTP incorporation?

A

Divalent atom e.g. mg2+ / monovalent

10
Q

Outline random primer labelling

A
  1. Linear dsDNA
  2. Denature + add random primer
  3. add unlabelled dNTP’s
  4. add labelled dNTP and klenow DNA polymerase
  5. release probe
11
Q

Nick translation for probe synthesis

A
  1. DNAse I introduces nicks to linear dsDNA
  2. DNAP I removes nucleotide using 5’-3’ exonuclease activity
  3. Labelled + unlabelled dNTP’s added
  4. Nucleotides replaced by 5’-3’ polymerase activity
  5. Denature for use as probe around 300 bp
12
Q

In vitro transcription for probe synthesis

A

cRNA ->

  1. linearise plasmid with page promoter + insert
  2. add A,C, G, UTP + DGUTP/ 32 p-UTP and polymerase
  3. RNA synthesis from phage promoter
13
Q

Oligonucleotide probe synthesis

A

T4 polynucleotide kinase with y-32p NTP (5’ end labelling) or terminal transferase with terminal transferase and either 3’ tailing or 3’end labelling

14
Q

DIG binds to UTP through

A

alkali soluble ether bond

15
Q

Why are probes hybridised?

A

To produce more labelled product

16
Q

What happens when we increase temperature during hybridisation?

A

Strand separation increase, ionic strength decrease

17
Q

Why is an oligonucleotide probe preferred to conventional?

A

Conventional is still stable after single mismatch which carries through to reduced hybridisation stringency so 20% mismatch in coding areas

18
Q

Light directed oligonucleotide synthesis can be either ___

A

A photochemical reaction or phorolitographic

19
Q

Photolithography involves ___

A

using glass which is hydroxylated and silonised so DNA an be covalently attached which determines which DNA can be exposed to light

20
Q

Autoradiogrphic detection

A

X-ray film/ phosphoimager

21
Q

Detection of non-radioactive probes:

A
  • colorimetric reaction
  • chemiluminescent reaction
  • fluorescent detection