Laboratory Isolation of Nucleic Acids 2 Flashcards
Is DNA stable or unstable
Often stable for long periods of time - when taken out of cells DNA is alot less stable and subject to degradation
Purified DNA is usually stable at 4 degrees C for atleast a year
Why should nucleic cids be quantified?
-ensure that the extraction process was effective
-ensure standardisation in further analysis of the DNA/RNA
-determine the concentration of nucleic acid relative to the concentration of protein that remains in the extracted sample
What are 2 methods of quantitation of nucleic acids?
-Electrophoresis - standard method for separating DNA
-Spectrophotometric analysis
What is electrophoresis?
The movement of charged molecules in an electric field, negatively charged molecules move towards the positive electrode and positively charged molecules move towrads the neg electrode
Wwhat are the 2 types of gels used in molecular biology?
-Agarose gels - easier to work with
-Polyacrylamide gels - higher resolution so can tell apart strands that are close in size
What is agarose and what size fragments can it separate?
-Agarose is a polysaccharide that forms gels with pores ranging from 100-300nm in diameter - size depending on conc of agarose - gel conc dtermines the range of DNA fragments that can be separated
-greater than 20kb - not resolved - will be a wedges band as cant fit through
-1-20kb - 0.8-1% gels
-less than 1kb - 2-3% gels
Why put comb at one end of gel for DNA separation?
Because DNA is neg charged so DNA has whole length of gel to separated towards the pos pole
What are the bands of DNA visualised with?
-Ethidium bromide (carcinogen) or sybr green staining either during or post electrophoresis
-fluorescent molecules that intercalates between bases
What is seen if high molecular weight genomic DNA is isolated?
-bright band with low mobility (near top of gel)
What will be seen in isolation of RNA
-2 distinct bands of rRNA (quality and ratio are important)
-2 - large subunit and small subunit
What is used to test DNA quantity and DNA quality
Electrophoresis - quality
Spectrophotometry - quantity?
What light absorbance is used for nucleic acids?
Nucleic acids absorb light at 260nm through the denine residues
-One OD unit at 260nm = 50mg/L (or 50µg/ml or 50ng/ul) DNA
-One OD unit at 260nm = 40ng/ul RNA
What do proteins absorb at?
Proteins absorb light at 280nm through the tryptophan residues
Quality of DNA ratios?
260nm/280nm = 1.6-2.0 good quality
260/280 = less than 1.6 = may be contaminated with protein
260/280 = greater than 2.0 = may be contaminated with RNA
How to get quantity and purity when given samples OD260 and OD280nm?
For quantity multiply the 260nm absorption reading by 50 (or 25 depending on wat OD)
For purity, compare 260 and 280 in a ratio
