Question

The purpose of the lysis procedure is:

to sequence the 16S rRNA gene

to separate the pieces of nucleic acid from one another

to break open the bacterial cells to release the genetic material

to amplify the 16S rRNA gene

Match the components of HotShot buffer with their role.

NaOH    

            removes anions so they cannot be used as cofactors by enzymes, increases pH to denature nucleases, disrupts lipid membranes, removes cations so they cannot be used as cofactors by enzymes, or decreases pH to denature nucleases

Detergent

            removes anions so they cannot be used as cofactors by enzymes, increases pH to denature nucleases, disrupts lipid membranes, removes cations so they cannot be used as cofactors by enzymes, or decreases pH to denature nucleases

Chelator

             (removes cations so they cannot be used as cofactors by enzymes)

When taking some of your bacterial isolate to place in the HotShot buffer you should take as much as can fit on the end of the pipette tip.

True OR False

The role of neutralization buffer is to:

restore the pH to approximately neutral

increase the pH to 10

introduce nucleases into the reaction

decrease the pH to 3

Nuclease-free water is added to the reaction mix to _____________________. Choose all that apply.

increase the volume of the solution without introducing nucleases which could degrade the nucleic acids

dilute the lysate so it is not too concentrated for later procedures

to stop the chemical reactions occuring in the solution

add nucleases to the reaction mix

Answer 1

1.

• DNA containing the target gene is isolated from specific cells. Thus, the cells needs to be lysed.
• The cell lysis solution may contain components such that the cellular components with the nucleic acid comes out.
• Answer: To break open the bacterial cells to release the genetic material

2.  

• Chelating agents like EGTA (Ethylene Glycol Tetraacetic acid) or EDTA (Ethylene diamine tetraacetic acid).
• Detergents like SDS(Sodium Dodecyl Sulfate) or lysozyme. This solution ruptures the cel.
• NaOH helps in cell lysis, raising pH to 12, denaturing proteins, and breaks hydrogen bond of nucleic acids to separate them.

• NaOH: increases pH to denature nucleases,

  Detergent: disrupts lipid membranes

  Chelator: removes cations so they cannot be used as cofactors by enzymes.

3.

True

So that air gap is nor present

4.

• Due to addition of NaOH, the solution becomes alkaline. Thus, to neutralize the pH, neutralising buffer is added.
• Answer: restore the pH to approximately neutral

5.

To correct the volume of the mixture and to dilute the components. Nuclease free water does not contain nucleases that may degrade the DNA.

• increase the volume of the solution without introducing nucleases which could degrade the nucleic acids
• dilute the lysate so it is not too concentrated for later procedures