Molecular Cloning And Gene Manipulation Flashcards
Restriction enzymes
Blunt end
5’ overhang
3’ overhang
Genetic library prep
Genomic DNA from tissue
Digest so fragment fits in vector
For BAC
Ligation of inserts to prepare vector DNA
What do plasmid vectors contain
Ori
Antibiotic resistance gene
Restriction sites
Genomic library contains
Plasmid library
Phage library
A library of bacterial artificial chromosomes BAC clones
DNA secuencing
Denature ddDNA Add primer and DA(tagc)P Aliquot in 4 test tubes Add appropriate dideoxy NTP and DNA polymerase Run in matrix
Methods of labelling
Original- radioactively label primer
Common- Fluorescent dideoxy
What does ORF VERIFY
Gene family- southern blot
Expression-Northern blot
Transcription initiation site- DNA footprint and EMSA
EXON site - s1 nuclease mapping assay
How does agarose separate DNA or RNA
Charge to mass ratio
Newly synthesised DNA is either
Radioactively labeled (32p)
Or
Chemically DIG MODIFIED DTTP
Southern blotting
Completely digest using several REnzymes
Label DNA probe with 32p DCTP
Allow binding the wash
Using labelled DNA probes- hybridisation what happens
Ss DNA unbound washed away
Only dd radioactive DNA probes left and are visible on xray
cDNA how does it work
Isolate mRNA by oligo(dt) column
Produce cDNA using oligo(dt) primer and reverse transcriptase
DNA polymerase produces second strand
DdDNA lighted with linkers with a endonuclease restriction site
DNA foot printing what happens
End label with 32p Allow TF to bind Cleave at random Denature DNA, separate by size Xray
ESMA How does it work
Prepare radioactively labeled DNA frag containing TF binding site
Incubate with tissue extract
Run on polyacrylamide gel
X-ray
Retarded bands indicate protein binding to DNA
EMSA what does it do
Determine if the protein is capable of binding to a given DNA or RNA sequence
S1 nuclease mapping
Label DNA with 32p Denature DNA and hybridise with mRNA Digest ssnucleic acid by s1 nuclease Run on polyacrylamide gel X ray
Northern blot
Same as southern
RNA separated by size
Microarray what happens
Prepare a cDNA library and make a systemic assaying a solid surface representing 1 gene Extract RNA and convert to cDNA Label cDNA with fluorescent dye cy3 Hybridise Genes expressed are yellow cuz of dye
Rt pcr
Reverse transcribe PCR
mrna prepared then converted to cDNA
Then gel electrophoresis shortest DNA travels furthest
Qrt PCR WHAT MAKES IT DIFFERENT TO PCR
Uses fluorescent dyes
Allows to monitor amplification. In real time
Amplification measured exponentially
Western blot SDS PAGE
Proteins
Denatured by negative SDS CHARGE
Negative charge allows proteins to migrate
Speed = net charge to mass ratio
Gene approach to loss of function
Gene knock out
Gene approach to gain of function
Gene knock in
Dominant negative analysis
Over expression
Loss of function miss expression
mRNA injected into egg
At site different to normal
Detected with antisense probe labeled with DIG
Gene knock out
Wt 4 petals
Mutation results in flours having lots
This indicated te AGAMOUS TF is regulating petal formation
Gain of function Dominant negative (dn) approach
Dn competes with wt
Dominant suppresses wt gene
Dominant negative (dn) approach
Heterotrimeric g protein Galpha binds GTP
Galpha hydrolysed is inactive now
Inability to break down GTP
Constantly activated
