RT-PCR Flashcards
(45 cards)
What is Nanodrop (spectrophotometric) and how does it work?
Direct correlation between absorbance and concentration. Nucleic acids absorb at many wavelengths, but have peak absorbance at 260 nm. Amount of light absorbance can be used to determine the concentration of RNA or DNA. Beer-lambert law is only linear for absorbancies between 0.1-1.0 this means that concentrations between 10.0 ng/uL and 3700 ng/uL when using the nanodrop can be detected accurately. Samples outside of this range should be dried or diluted to produce more accurate results.
Interpreting Nanodrop (spectrophotometric) Results at 260 nm
Nucleic acids absorb UV light at 260nm due to aromatic bases within structure.
Purines (Thymine, cytosine, and uracil)
Pyrimidines (adenine and guanine)
all have absorbencies at 260nm.
Purines and absorbencies level with nanodrop
Thymine, cytosine, and uracil
260nm
Pyrimidines and absorbencies level with nanodrop
adenine and guanine
260nm
Interpreting Nanodrop (spectrophotometric) Results at 280 nm
Where proteins and phenolic compounds have strong absorbencies.
Aromatic amino acid side chains (trytophan, phenylamine, tyrosine, and histidine) within the proteins are responsible for this absorbancies.
Aromaticity of phenol groups or organic compounds absorb strongly at 280nm
Aromatic amino acid side chains absorbance peak on a nanodrop show at
280nm
Aromatic phenol groups absorbance peak on a nanodrop show at
280nm
Interpreting Nanodrop (spectrophotometric) Results at 230 nm
Many different organic compounds have strong absorbencies at 225nm.
Peptide bonds in proteins absorb light between 200-230 nm.
Peptide bonds in proteins absorbance peak on a nanodrop show at
200-230nm
A260/280 ratio Interpreting Nanodrop (spectrophotometric) Results
This is used to determine the protein contamination of a nucleic acid sample.
Aromatic proteins have a strong UV absorbance at 280nm.
For pure RNA and DNA A260/280 ratios should be somewhere around 2.1 and 1.8.
A lower ratio indicates that the sample is protein contaminated. This can have an impact on the down stream applications that use the nucleic samples.
A260/230 ratio Interpreting Nanodrop (spectrophotometric) Results
This is used to indicate the presence of organic contaminants (phenol, TRIzol, salts, and aromatic compounds).
Samples with 260/230 ratios below 1.8 have a significant amount of these contaminants and this could interfere with downstream applications.
Especially Reverse Transcription.
The ratio should be close to 2.0.
Gel interpretation:
If the gel looks the same at all wells it is _____.
contaminated
Gel interpretation:
+ control is ________.
a sample that is guaranteed to work, such as a DNA ladder.
Gel interpretation:
- control is ________.
a sample where the component is omitted so that the sample/procedure fails. We do not put in the plant/DNA in, but we keep all the reagents of the mix/solution.
Gel interpretation:
Quality
the gel is dependent on the distance that the sample DNA/RNA travels. The farther the better.
Gel interpretation:
Quantity
This accessed with the nanodrop.
If you run a RNA gel, but see large chromosomes, this tells you that your sample is polluted with DNA.
If a Gel (RNA) is fuzzy without tell tale signs of rRNA bands then it is most likely _______.
Contaminated by RNase damage.
How does gel differ between RNA and DNA.
They have different bp.
For RNA Gel DNA is removed by a _____.
DNase enzyme, but it does not degrade RNA.
A perfect RNA gel sample will show _____.
small and large subunits from a total RNA extraction. There will also be faint smears representing the thousand of differently sized mRNA.
Agarose gel electrophoresis is used because _____.
greater range of separation. Small DNA fragments (50-20,000 bp) are best resolved in agarose gels.
Used to separate, identify, and purify DNA fragments.
Factors that affect gel Electrophoresis gels:
- composition and concentration of the buffer
- concentration of agarose gel
- purity and concentration of DNA
- use of the buffer and agarose gel
- preparation of the gel
- pH of the buffer and DNA
- angles between the two electric fields
- relative strength of the electric field
- length of the electric pulses
Gel charge on the wells to the bottom is _____.
negative (wells) to positive.
Total DNA
nucleus, mitochondria, and cholorplast
