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Custom Genomics > Sample QC > Flashcards

Sample QC Flashcards

1
Q

how does the nano drop work?

A

its a spectrophotometer that measures the light absorbed by a solution: Absorbance

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2
Q

what information does the Nanodrop provide you with?

A
  • Purity, DNA/RNA (contaminations)

- concentration (ng/uL)

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3
Q

what information can’t the Nanodrop provide?

A
  • integrity of the sample (Degradation)

- can’t tell if RNA id contaminated with DNA and vice versa

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4
Q

what does the absorbance ratio at 260/280nm tell you

A

highlights the possible contamination by proteins

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5
Q

what does the absorbance ratio at 260/230nm tell you

A

highlights the possible contamination by salt, carbs, peptides and phenol

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6
Q

what is considered the optimal ratio for DNA and RNA at 260/280nm

A

DNA 1.8 - 2.0

RNA 2.0 - 2.2

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7
Q

what is considered the optimal ratio for DNA and RNA at 260/230nm

A

DNA 2.0-2.2

RNA 2.0 - 2.2

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8
Q

what is the reason for the optimal ratio of DNA being lower than that of RNA at 260/280

A

because uracil has a higher absorbance ratio than thymine when measured individually causing the base absorbance of RNA to be naturally slightly higher than that of DNA

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9
Q

at what wavelength does protein absorb UV light

A

280nm

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10
Q

at what wavelength do salts, carbs, peptides and phenol absorb UV light

A

230nm

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11
Q

what is the concentration range of the nano drop

A

25-2000ng/uL

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12
Q

what equation is used to convert A260 values into ng/uL

A

beer-lambert law

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13
Q

how can a sample be determined to be DNA or RNA using the A260 value and the beer-lambert law

A

when A260 = 1 then:

  • the constant will be 50 if its DNA
  • the constant will be 40 if its RNA
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14
Q

On the absorbance plot for the nano drop, what relationship should the 280 and 230 peaks have with the 260 peak

A

the 280 and 230 peak should be half the hight of the 260 peak

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15
Q

what wavelength do DNA and RNA absorb UV light at

A

260nm

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16
Q

what are the benefits of using nanodrop

A

quick and affordable

only needs small amount of sample i.e. 25ng/uL

17
Q

how is the RIN calculated

A

the ratio created by putting the 28S peak over the 18S peak

18
Q

why is nano drop so important when QCing in studies

A

its the only way to assess purity and contamination in DNA and RNA

19
Q

what is the Qubit

A

a quantification method that uses fluorescent dyes that bind to DNA/RNA molecules which then emit light when excited

20
Q

what is the concentration range of the Qubit

A

DNA 2-1000ng/uL

RNA 20-1000ng/uL

21
Q

how is the qubit sample prepared

A

mixed with the dye and kit buffer in an eppendorf

22
Q

what information is gained from the Qubit

A

sample concentration

- fluorescence values converted into conc,

23
Q

benefits of Qubit?

A
  • more sensitive and accurate

- good for PCR and NGS as it already contains the fluorescent tags to be amplified

24
Q

what information is not provided by Qubit

A

no info on purity

no info on integrity

25
Q

what is the mechanism of the tapestation

A

it performs automated electrophoresis in an agarose gel which separates molecules based on their size and charge using an electrically charged field

26
Q

what charge do DNA and RNA molecules have

A

negative

27
Q

what constitutes the RNA analysis

A

RNA ladder
RNA buffer
RNA sample
RNA screentape

28
Q

what is the RNA ladder

A

a set of different and known RNAs band size used as a reference for estimating size and mass of an RNA aample

29
Q

what type of RNA is used as the assessment of RNA

A

Ribosomal RNA (rRNA)

30
Q

what is the RIN

A

A numerical value between 1 and 10 used to indicate RNA integrity

31
Q

what is the threshold RIN value for an RNA sample to be deemed to have high enough integrity and suitable for downstream applications?

A

7

32
Q

how are quantity and integrity derived form the tapestation/bioanalyzer graph

A

concentration is derived from the height of the peak (quantity)
size of fragments is based on width of the peak (integrity)

33
Q

what information does tapestation/bioanalzyer give you

A
  • integrity of DNA/RNA
  • Info on size, quantity and quality
  • shows whether there is a DNA or RNA contamination
34
Q

what information about contamination can the bioanalyzer tell you that nano drop can’t?

A

whether DNA is contaminated with RNA and vice versa

35
Q

what information can’t the bioanalyzer tell you

A

no information about purity other than DNA and RNA contaminations

36
Q

why is the tapestation better than other integrity analysis methods like gel electrophoresis?

A

because gel electrophoresis only allows you to visually assess purity however tapestation offers an objective RIN value that uses a standardised equation/procedure.

37
Q

how is DNA concentration determined using the bioanalyzer graph

A

using the area under the peak compared to the known conc of the upper marker

Custom Genomics (4 decks)

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