NA Isolation and Purification Flashcards
Common practices prior to isolation
- RNase-/DNase-free reagents, plasticware, and glassware
- sterilization
- surface decontamination
General Steps in NA Isolation
- tissue homogenization or cell lysis
- denaturation and separation of NA from other biomolecules
- NA precipitation
- washing of precipitated NA
- drying of pellet and dissolution of dried pellet
a denaturant of proteins that disrupts non-covalent interactions and destroys the molecular structure
guanidium (guanidine isothiocyanate)
Common chemicals used for extraction
- buffer
- salt
- cell lysis reagents
- denaturants
- reducing agents
Common alcohol used in NA precipitation
EtOH (95% to absolute)
isopropanol
T or F: when washing precipitated DNA/RNA, you can also use 95% ethanol
False. You use 70-80% ethanol
After NA isolation, if you want only the DNA, what do you treat your pellet with?
RNase
After NA isolation, if you want only the RNA, what do you treat your pellet with?
DNase
Why not incorporate RNase/DNase in the earlier protocol steps of the isolation?
Because these reagents are expensive so you want to estimate how much treatment you will incorporate relative to the amount of the your product.
What happens after RNase/DNase treatment?
You should repeat isolation from steps 2-5, denature and separate, precipitate, wash, dry, and dissolve.
commonly used in checking DNA quality
native agarose gel electrophoresis
commonly used in checking RNA quality
denaturing gel electrophoresis (with formaldehyde and MOPS buffer)l
When do you use denaturing gel electrophoresis?
When you will subject your sample to RE digestion or sequencing.
What is the function of formaldehyde and MOPS in the denaturing gel electrophoresis?
It maintains the linear structure of the RNA, preventing from formation of secondary structures or RNA looping
determination of DNA concentration and purity (4)
- gel electrophoresis
- spectrophotometerr
- fluorometer
- microfluidics
maximum absorbance of light for DNA
A260
maximum absorbance of light for proteins
A280
if A260/280 ratio is more than 2.0
contaminated with organic compounds such as phenol and chloroform
if A260/280 ratio is less than 1.8 (or 1.6)
protein contamination
ideal A260/280 ratio
1.8-2.0
T or F: For it to be a good DNA/RNA, A260/A280 < A260/A230
True
Calculate concentration of DNA
= A260 x DF x 50ug/mL
calculate concentration of RNA
= A260 x DF x 40ug/mL
calculate yield
yield = concentration x total remaining volume
