Nucleic Acid Quantification & Purity Flashcards
(24 cards)
What is the primary purpose of nucleic acid quantification?
Assessing DNA & RNA concentration and purity before downstream applications like PCR, qPCR, sequencing, and cloning.
What is the most common method for nucleic acid quantification?
Absorbance-based quantification using UV spectrophotometry (NanoDrop).
What method offers higher specificity in nucleic acid quantification?
Fluorescence-based quantification (e.g., Qubit).
What is the simplest method to assess nucleic acid purity?
Measuring absorbance ratios at specific wavelengths.
What is the ideal A260/A280 ratio for pure DNA?
> 1.8
What is the ideal A260/A280 ratio for pure RNA?
~2.0
What does the A260/A230 ratio assess?
Organic & salt contamination.
What is the ideal A260/A230 ratio for a pure sample?
~2.0
How does protein contamination affect the A260/A280 ratio?
It lowers the ratio (<1.8).
What effect do carbohydrates have on the A260/A280 ratio?
They lower the ratio.
What contaminants do not significantly affect the A260/A280 ratio?
Organic compounds and salts.
What are the two main types of quantification methods?
- Absorbance-Based Methods (e.g., NanoDrop, UV Spectrophotometry)
- Fluorescence-Based Methods (e.g., Qubit, SYBR Green, PicoGreen)
What is a disadvantage of absorbance-based methods?
Cannot distinguish DNA from RNA.
What is a key advantage of fluorescence-based methods?
Higher sensitivity & specificity than absorbance methods.
What is a possible cause of a low A260/A280 ratio?
Protein contamination.
What solution can improve low A260/A280 ratios?
Improve protein removal, use additional washes.
What could cause a low A260/A230 ratio?
Salt or organic contamination.
What action can be taken to resolve low A260/A230 ratios?
Additional ethanol washes, adjust purification method.
What does a high A260/A280 ratio indicate?
RNA contamination in DNA prep.
What can be done to treat RNA contamination in DNA preparations?
Treat with DNase-free RNase.
What is the key takeaway regarding quantification methods?
Absorbance-based (NanoDrop) & fluorescence-based (Qubit) quantification are key methods.
Fill in the blank: A260/A280 ratios greater than 1.8 for DNA and approximately 2.0 for RNA ensure _______.
protein-free nucleic acids.
Fill in the blank: A260/A230 around 2.0 confirms _______.
low salt & organic contamination.
True or False: Fluorescence-based methods are less specific than absorbance-based methods.
False.
