Zusammenfassung der Ressource
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Electroporations- passing electricity through a mix of DNA and bacteria causes DNA to be taken up
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What does N stand for?
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What are the meanings of i and g?
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i = Library size
g= probability of getting desired gene
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i = avg insert size
g = genome size
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i = probability of getting desired gene
g = genome size
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The larger the genome, the larger the library required to replicate it
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Converted from mRNA
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Converted from snoRNA
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Converted from siRNA
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Less than 1% of the genome is mRNA, but it's exclusively expressible genes
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What is required to make the first RNA-DNA hybrid?
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dNTP's
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Primer
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Reverse Transcriptase
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What are the two methods of degrading the RNA part?
Frage 10
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Which method was used to get the following DNA strand?
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Alkali degrading
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Boiling
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Shaking
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RNAase H
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What is required to get from the former to the latter?
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dNTP's
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DNA Polymerase
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Primer
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Reverse transcriptase
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What method was used to degrade the RNA into the following?
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RNAase H
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Alkylation
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Boiling
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Sonication
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What is needed to get from the former to the latter?
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dNTP's
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DNA Polymerase H
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RNAase
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rNTP's
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When creating cDNA, a little information is always lost from the 3' end
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How do we prevent this loss of information?
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After degrading the RNA, add a homopolymer (polyC) tail to the 3 end
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Using Terminal Transferase
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Before degrading the DNA, degrade the Poly A tail
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Then in second strand synthesis add ...
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dNTP's
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DNA polymerase
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Oligo-dG primer
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Enriching desired DNA means creating/finding more of it
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Selective Hybridisation removes cDNA's common to more than one tissue
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Tissue Specificity is useful if the gene is only expressed in a certain tissue
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What is Normalisation?
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Melt, anneal and isolate ssDNA
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Use restriction enzymes to repeatdely cut and ligate DNA
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Use DNA helicase to continually replicate DNA
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How does Normalisation work?