Electroporations- passing electricity through a mix of DNA and bacteria causes DNA to be taken up
What does N stand for?
The no of clones needed
P of getting a clone with the desired gene
Genome size
What are the meanings of i and g?
i = Library size g= probability of getting desired gene
i = avg insert size g = genome size
i = probability of getting desired gene g = genome size
The larger the genome, the larger the library required to replicate it
When is cDNA used?
When an organism has a lot of non-coding DNA
When an organism is very small
When the DNA is degraded
How is cDNA made?
Converted from mRNA
Converted from snoRNA
Converted from siRNA
Less than 1% of the genome is mRNA, but it's exclusively expressible genes
What is required to make the first RNA-DNA hybrid?
dNTP's
Primer
Reverse Transcriptase
What are the two methods of degrading the RNA part?
Alkylisation (RNA is less stable)
RNA digestion through RNAase H
Boiling
Restriction enzymes
Which method was used to get the following DNA strand?
Alkali degrading
Shaking
RNAase H
What is required to get from the former to the latter?
DNA Polymerase
Reverse transcriptase
What method was used to degrade the RNA into the following?
Alkylation
Sonication
What is needed to get from the former to the latter?
DNA Polymerase H
RNAase
rNTP's
When creating cDNA, a little information is always lost from the 3' end
How do we prevent this loss of information?
After degrading the RNA, add a homopolymer (polyC) tail to the 3 end
Using Terminal Transferase
Before degrading the DNA, degrade the Poly A tail
Then in second strand synthesis add ...
DNA polymerase
Oligo-dG primer
Enriching desired DNA means creating/finding more of it
Selective Hybridisation removes cDNA's common to more than one tissue
Tissue Specificity is useful if the gene is only expressed in a certain tissue
What is Normalisation?
Melt, anneal and isolate ssDNA
Use restriction enzymes to repeatdely cut and ligate DNA
Use DNA helicase to continually replicate DNA
How does Normalisation work?
Repetitive DNA binds to other repetitive DNA
Leaving only unique DNA single stranded
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