exam 2 Flashcards
What is the purpose of mechanical grinding in plant DNA extraction?
Breaking the cell walls
What is the function of using a detergent in DNA extraction? What is a common one used?
To disrupt the cell membranes. Most common is SDS (sodium dodecyl sulfate)
What is the function of EDTA in DNA extraction?
Chelates heavy metals like Mg to remove metal cofactors from proteases and nucleases which would degrade DNA. Protect from endogenous nucleases
Why do we limit time in DNA extraction?
To prevent degradation
What is the purpose of a buffering agent in DNA extraction? Why is it important in plant DNA extraction? What is an example?
Maintains pH since plant vacuole is large and acidic. Example is Tris
What is the purpose of beads in DNA isolation?
Grind the tissue of your sample
What are the main components in a PCR reaction?
Master mix, forward/reverse primer, nuclease free water, DNA template/control group
What is included in a master mix (4)?
dNTPs, Mg2+ ions, buffer, taq polymerase
What are the components of a traditional PCR reaction (7)?
Template DNA, Taq polymerase, dNTPs, forward primer, reverse primer, buffer (Mg2+, Tris buffer), water
How long is the extension step? At what temp?
3-6 minutes at 72ºC
How long is the denaturation step? At what temperature?
2-3 minutes at 95ºC
How long is the annealing step? At what temperature?
30 seconds, 45-65ºC
What happens in the repeated cycles during PCR?
95ºC- 30 sec- denaturation
45-65ºC- 30 sec- annealing
72ºC- time varies- extension
How do you determine how long the extension step should be based on kB length?
1 minute per kB
What precipitates out RNA in plant DNA isolation?
LiCl
What solubilizes membranes in plant DNA isolation?
SDS
What controls the pH of the extraction in plant DNA isolation?
Tris-Hcl
What binds divalent metal ions like Mg++, inactivating nucleases in plant DNA isolation?
EDTA
What precipitates DNA out of the solution of cell contents in plant DNA isolation?
Isoproponal
What removes cell debris in plant DNA isolation?
Centrifugation
What binds to divalent metal ions, like Mg++ to inactivate nucleases in human mtDNA isolation?
Chelex
What denatures protein and RNA in human mtDNA isolation?
High heat
What maintains the pH of the extraction in human mtDNA isolation?
Tris-HCl
What removes cell debris in human mtDNA isolation?
Centrifugation
What components of plants can affect purification (2)?
Contaminants or secondary metabolites
What are some considerations when it comes to purifying DNA (4)?
-neutralizing and avoiding DNA degradation and DNA degrading enzymes
-removing DNA from the natural barriers of the cell
-purifying DNA away from other nucleic acids such as RNA
-purifying DNA away from other contaminants or secondary metabolites
Which direction does DNA synthesis occur in?
5’ to 3’
How can you determine SNP variability?
Sequencing data from PCR product
Can agarose gel electrophoresis indicate DNA sequence differences?
No, only band size
What does “cleaning up” the PCR product mean?
Removing residual template DNA, proteins, or primers
What happens if you don’t clean up the DNA?
Contaminates PCR product and inhibits sequencing reaction
What concentration of DNA is sent for sequencing?
100ng
What is included in sequencing reactions?
DNA, water, and primer
What volumes change when sequencing?
Water and sample volumes, company dependent
How many primers will be included in one tube to be sent to sequence?
One, either forward or reverse
What does DNA polymerase require for elongation? What is its purpose?
3’-OH. Catalyzes the formation of a new phosphodiester bond.
What provides the 3’-OH for DNA polymerase?
Primers
What is a positive control?
Confirms that your assay or experiment worked. Gives a parallel comparison.
What is a negative control?
Confirms you are not getting false positive results. Demonstrates what a negative result looks like
What are the two most variable components in a PCR reaction?
DNA template/ control group and primers
Where is DNA in the clean up protocol?
Spin column
What volume of membrane binding solution is used to clean PCR amplification?
Equal volume of PCR amplification. 20uL of PCR = 20uL of membrane binding solution
What is elution in PCR purification?
DNA purification from the spin column. Done with water and centrifuge.
