What is the purpose of Proteinases in DNA extraction?
Proteinase K is used during DNA extraction to digest many contaminating proteins present. It also degrades nucleases that may be present in DNA extraction and protects the nucleic acids from nuclease attack.
What does phenol-chloroform do in DNA extraction?
The main function of chloroform is to protect genomic DNA during a catastrophe. Chloroform increases the efficiency of phenol to denature the protein. Here, chloroform allows proper separation of the organic phase and aqueous phase and keeps DNA protected into the aqueous phase.
How does the Chelex extraction method work?
Principle: Chelex resin works by preventing DNA degradation from degradative enzymes (DNases) and from potential contaminants that might inhibit downstream analyses. In general, the Chelex resin will trap such contaminants, leaving DNA in solution.
What is the principle of DNA and RNA extraction?
The basic principle of the method is the separation of RNA from DNA and proteins after extraction with an acidic solution, which consists mainly of GuSCN, sodium acetate, phenol, and chloroform, followed by centrifugation.
Why do we use chloroform in RNA extraction?
Chloroform is one important reagent for RNA purification with guanidinium thiocyanate-phenol-chloroform extraction method. It is used to promote phase separation so that RNA is isolated from DNA and proteins in a biological sample.
How does chloroform promote phase separation?
Because the phenol:chloroform mixture is immiscible with water, the centrifuge will cause two distinct phases to form: an upper aqueous phase, and a lower organic phase. The aqueous phase rises to the top because it is less dense than the organic phase containing the phenol:chloroform.
Why is Chelex used in PCR?
Chelex can chelate a large amount of divalent ions that may be donated by the sample, and the Chelex beads can be easily removed so that they will not interfere with subsequent PCR amplifications that require Mg++.
What is Chelex made of?
Chelex 100 Resin is made of a styrene divinylbenzene copolymer containing paired iminodiacetate ions, which act as chelating groups in binding polyvalent metal ions.
How is EDTA removed from DNA?
Just mix 20 mM EDTA with your ethanol at used proportions and centrifugate after about 20 min to see if there is any precipitate. Dear Georgi, I did not know how you extracted your DNA, but EDTA is rarely could be problem because it easily can be removed by washing the DNA by 70% EtOH.