Week 3 - Purifying DNA and RNA Flashcards
List the general steps for DNA preparation
- centrifuge a culture
- cells are removed and broken to give a cell extract
- DNA is purified from cell extract
- Concentrated DNA
What techniques can be used to release DNA from cell
Mechanical methods:
- Glass beads, sonication
Chemical methods:
- Solvents, enzymes, detergents, denaturants
MM lyse cell walls but could damage DNA
CM is gentler in contrast
How can DNA be purified from contaminants?
with the use of enzymes like RNase and proteases, precipitation with phenol or CTAB. Ion exchange chromatography.
RNase, Proteinase K, Phenol with CHCI3 are all methods of contaminant removal (purification) describe their methods of action
RNase enzymatically removes RNA, but must be DNase free
Proteinase K enzymatically romves proteins.
While Phenol denatures proteins which can be removed after centrifugation, Phenol must be a 1:1 mix with the extract. Addition of CHCI3 then removes any of the remaining Phenol.
Explain the process of Silica binding
Denaturant is used for proteins, while DNA binds to silica resin, contaminants are exclued as they bind less effectively or not at all, pure DNA is elted by washing in a salt free buffer (WATER)
How can DNA concentration by increased after purification
Via precipitation with ethanol or isopropanol in the presence of salt
How is Plasmid DNA purified?
Plasmid DNA must be separated from chromosomal DNA, This is done by lysing the cells, sample should then be centrifuged to separate
3 states of DNA in cells
Supercoiled (natural state), Circular and Nicked (Open)
How is DNA purity measured?
The sample is measured at A260 and A280, say A260 gives 1.4 and A280 give 0.75 the formula is as follows:
1.4/0.75 = 1.87
A ratio of less then 1.8 indicated a contaminated sample
List some differences of RNA to DNA
RNA is chemically similar to DNA
RNA molecules are shorter than genomic DNA
RNA is highly abundant in cells
RNA has a short half life in cells
Is biologically unstable
RNA is chemically stable
List the estimated quantities of DNA RNA and proteins per cell
DNA = 0.73 pg
RNA = 0.48 pg
Proteins = 4.32 pg
List RNA types in eukaryotic cells
hnRNA - pre-mRNA
ncRNA
miRNA - mitochondrial
tRNA - Highest quantities in cells
rRNA
mRNA - heaviest molecule
Inactivating RNases why and how?
RNases destroy RNA and are present in cells and tissues and need to dealt with, methods include:
Denaturant
high concentrations of ammonium sulphate
Reagents such as RNAsin (RNase inhibitor)
Chemical treatments - DEPC, DMPC
What are Oligonucleotides?
Short ss or ds DNA molecules
chemically synthesised and highly purified
15-60 bases
non-phosphorylated
used to make PCR primers, adaptors, probes, linkers, whole genes
