Enzymes and Primers Flashcards
Why is CIP used in plasmid cloning?
Removes 5’ and 3’ phosphate of linear DNA to prevent religation with speed and high specificity.
Problems with CIP
- Difficult to eliminate via heat denaturation
- Sticks to DNA ends
- If residual CIP carries through to ligation, insert will be dephosphorylated
- Too much will damage DNA
Why is SAP a good alternative to CIP?
Rapid irreversible heat inactivation at 65C
Polymerase I was orinigally purified in _____ but ubiquitous in ______ year _____
E.coli, prokaryotes, 1956
How is the klenow fragment made?
Domain necessary to code for 5’-3’ exonuclease activity removed by proteolytic treatment of PolI
Problems with using klenow
New enzyme would have to be added every cycle
Taq can be used for ___ min at ___ or ____ at ___
30 , 95, 9, 97.5
Problem with Taq
no 3’-5’ exonuclease activity
What does Pol I need for dNTP incorporation?
Divalent atom e.g. mg2+ / monovalent
Outline random primer labelling
- Linear dsDNA
- Denature + add random primer
- add unlabelled dNTP’s
- add labelled dNTP and klenow DNA polymerase
- release probe
Nick translation for probe synthesis
- DNAse I introduces nicks to linear dsDNA
- DNAP I removes nucleotide using 5’-3’ exonuclease activity
- Labelled + unlabelled dNTP’s added
- Nucleotides replaced by 5’-3’ polymerase activity
- Denature for use as probe around 300 bp
In vitro transcription for probe synthesis
cRNA ->
- linearise plasmid with page promoter + insert
- add A,C, G, UTP + DGUTP/ 32 p-UTP and polymerase
- RNA synthesis from phage promoter
Oligonucleotide probe synthesis
T4 polynucleotide kinase with y-32p NTP (5’ end labelling) or terminal transferase with terminal transferase and either 3’ tailing or 3’end labelling
DIG binds to UTP through
alkali soluble ether bond
Why are probes hybridised?
To produce more labelled product
