Nucleic acid extraction Flashcards
The first step of nucleic acid extraction is to disrupt the tissue/cell => produce a lysate, how is this done (explain)?
- Protease (Protinase K)
- Detergents (SDS): break membrane
- tissue homogeniser
- Chaotrophic salts: disrupt structure of proteins & NA
- Phenol: only for RNA isolation
Describe the eneral process of NA extraction
- Produce lysate
- Separate soluble (NA) & insoluble (cell debris, fat) from lysate
- Purify NA sample
2 types of purifying methods
- phenol-chloroform
- Comlumn-based methods
Summarise the steps for phenol-chloroform extraction
- equal vol. of phen-chlor mixture added to lysate & centrifuged (mix)
- => upper aqueous phase separated from lower organic phase
- add sodium acetate?
- precipitate NA by adding isopropanol
- collect NA by centrifugation & wash w/ ethanol
- let EtOH evaporate
- Resuspend NA w/ Tris-EDTA
which phase do NA & proteins lie after phen-chloro is added ? & why?
Aq: NA bc phosphate back bone = neg. charge = polar
Org: proteins bc hydrophobic core - interact w/ phenol
Interface: hydrophilic regions of proteins
What happens when ph of phenol is acidic (~5 ie. 5:1 Ph-Ch) or basic (~8 ie. 1:1)
A: DNA in org bc neg charge @ backbone neutralised w/ H+ ions. RNA in org bc dNTP on ss can form H bond w/ water
B: DNA & RNA = neg charge = polar = aqu.
How is NA precipitated out of Aq. phase? *(mol gen lab manual)
DNA is polar & alcohol (isopropanol) is non-polar = NA is insoluble & precipitate out of solution
Summarise the steps for column-based extraction method
- Sample is lysed => lysate
- alcohol & lysate added in column
- NA binds to silica in membrane
- column is washed w/ chaotrophic salt -> alcohol (remove salt) & dryspun (remove residual alcohol)
List adv. & disadv of Ph-Ch method & C-B method
- Ph-Ch: √ inexpensive; separate DNA from RNA. X Labourious; handling of hazardous chemicals
- C-B: √ Fast, safe, easy, can be automated. X expensive?
Complications in RNA extraction?
- RNA less stable than DNA bc ss & hydrolysed @ basic pH
* RNase difficult to remove = must take extra precautions to keep equip. & reagents RNase-free
Equ to find quanity of NA
[NA] = OD260 x Standard coefficient *(Std)DNA = 50 ug/ml (Std)RNA = 40 ug/ml
Equ. to determine purity of NA sample (NA vs protein). & interpret results
260/280 ratio = OD260 / OD280
* NA PURE if ratio b/w 1.8 - 2.0
Depending on the method used, purified RNA
contains all subtypes of RNA (what are they?)
tRNA, mRNA, rRNA
We are most interested in ___________ RNA,
which may not be visible on the gel
mRNA
Instead, the _______________ RNA is used to
assess the integrity of purified RNA
rRNA
