Latest topics
» Quiz Time/1
Sun Nov 22, 2015 10:24 pm by mithu04

» Quiz Time/1
Sun Nov 22, 2015 10:20 pm by mithu04

» Quiz time/2
Sun Nov 22, 2015 10:08 pm by mithu04

» Equipment maintenance
Sun Nov 22, 2015 10:07 pm by mithu04

» Quiz time/ 05
Sun Nov 22, 2015 10:05 pm by mithu04

» Quiz time/ 04
Sun Nov 22, 2015 9:59 pm by mithu04

» Quiz time/ 03
Sun Nov 22, 2015 9:58 pm by mithu04

» Quiz time/ 02
Sun Nov 22, 2015 9:56 pm by mithu04

» Quiz time/ 01
Sun Nov 22, 2015 9:55 pm by mithu04


Quiz time/ 01

View previous topic View next topic Go down

Quiz time/ 01

Post by Admin on Thu Oct 08, 2015 2:10 pm

Whole blood DNA extraction - Answer the following in brief and explain where necessary.

1. Explain the main principles underlying whole blood DNA extractions in brief.

2. Why is 70% ethanol added to precipitate DNA?

3. What is a chealating agent? In which steps are chealating agents used in blood DNA extraction?

4. What is an elution buffer?

5.In which step is 100% isopropanol added?

6. What is the purpose of using 100% ethanol?
avatar
Admin
Admin

Posts : 28
Reputation : 0
Join date : 2015-05-16

View user profile http://irdgenetics.forumotion.me

Back to top Go down

Re: Quiz time/ 01

Post by Ravindee on Sat Nov 21, 2015 3:51 pm

1) The main components of whole blood are RBC's , WBC's and platelts. Basic steps in any type of DNA extraction includes; cell lyis, DNA precipitation, washing step and resuspension step.

Cell lysis step: First we should get rid of RBC's and platelts because, what we need is WBC's. why becuase WBC's has nuclei and inside the nucleus you can find DNA.So the cell lysis step includes getting rid of unnecessary blood components and also breaking of cell membrane. This is achieved thru the use of detergents and enzymes. They solubilize cell membranes and also enzymes combine with detergents to target cell surface or cytosolic components.

Next step is DNA precipitation: By adding high concentration of salts to DNA containing solution, as cations from salts such as ammonium acetate counteract repulsion caused by the negative charge of the phosphate backbone. A mixture of DNA and salts in the presence of solvents like ethanol or isopropanol causes nucleic acids to precipitate.

In the washing step we use 70% ethanol to wash away the alcohol remnants and other contaminants like salts.

In the final step, i.e the resuspending step, we resuspend in water or TE buffer for long term storage because it prevents it from being damaged by nucleases, inadequate pH, heavy metals and oxidation by free radicals.
avatar
Ravindee

Posts : 19
Reputation : 15
Join date : 2015-09-17

View user profile

Back to top Go down

Re: Quiz time/ 01

Post by Ravindee on Sat Nov 21, 2015 3:59 pm

2)DNA is washed with 70% ethanol to remove some of the pellet. If water was used as the wash then DNA would dissolve again and if 100% ethanol was used the salt would not wash off because the sodium salts are partially soluble in ethanol. Use of 100% ethanol causes complete removal of water from the DNA & eventually denatures DNA molecules. 70% is the optimum percentage for the salts to wash away remaining DNA.

avatar
Ravindee

Posts : 19
Reputation : 15
Join date : 2015-09-17

View user profile

Back to top Go down

Re: Quiz time/ 01

Post by Ravindee on Sat Nov 21, 2015 4:22 pm

3) A chelating agent is a substance whose molecules can form several bonds to a single metal ion. In other words, a chelating agent is a multidentate ligand. It is used to sequester divalent cations, such as Mg2+ and Ca2+, which prevents enzymes like DNase from degrading the DNA.

It is used in the Lysis step or extraction step.

4)Elution buffer is a major solvent in affinity chromatography. Elution buffer is used to wash away unbound proteins at first and at a greater concentration it releases the desired protein from the ligand.

5)100% isopropanol is added in the DNA precipitation step.

6)salts are generally less soluble in isopropanol than in ethanol, they have more of a tendancy to co-precipitate with the DNA. So to lessen the chances of salt precipitation, isopropanol precipitations are carried our at room temperature with minimal incubation times.
avatar
Ravindee

Posts : 19
Reputation : 15
Join date : 2015-09-17

View user profile

Back to top Go down

Re: Quiz time/ 01

Post by Sponsored content


Sponsored content


Back to top Go down

View previous topic View next topic Back to top

- Similar topics

 
Permissions in this forum:
You cannot reply to topics in this forum