Exam 2 Flashcards
One of the first steps of a DNA extraction involves a mortar and a pestle, what is this step?
Grinding
A buffer containing enzymes is used to “open the cells” during incubation. This process is known as?
Lysis
The amount of enzymes used in a reaction affects the efficiency of the enzymes activity. Name 3 other factors that affect enzyme activity.
Temperature, pH, and time
After lysing the cell open, the sample is centrifuged after which the DNA is located where in the tube?
Precipitant
When extracting DNA, the precipitate is added to the QiAshredder and centrifuged. What happens to the DNA during this step?
Binds to the column
After isolating DNA from the gel, a gel electrophoresis was ran for the purpose of ___ the DNA.
Analyzing
The purpose for washing a column containing bound DNA is?
Washing away impurities
After the last column wash, the DNA is removed from the column by the process of?
Eluting
Name 3 reagents that are needed in a PCR MasterMix for a successful reaction to occur.
GoTaq, forward primer, reverse primer
Cells that can easily incorporate DNA from their environment are called __ cells.
Competent
In a PCR reaction, what is responsible for replicating the DNA?
Forward and reverse primers
Primers consist of ___.
Oligonucleotides
Name a DNA polymerase used in a PCR that does not have proofreading abilities.
Taq polymerase
There are three primary temperature changes in PCR reaction:
Denaturation at 94C
Annealing at 54C
Elongation at 72C
The temperature a PCR product should be stored at is?
4C
Name the machine that we used to run our PCR in.
Thermocycler
What does PCR stand for?
Polymerase chain reaction
Name the process of using electricity to run DNA through a substance such as an agarose matrix.
Gel electrophoresis
The PCR products were loaded onto a ___ to determine if the DNA was present or absent.
Agarose gel
After amplifying extracted plant DNA by PCR, and after running the PCR produce through a gel; how was the DNA removed from said gel?
Excision
A “ladder” is also known as..
Gene ruler or molecular ruler
DNA shows up on a gel in the form of a ___.
Band
Absorption of DNA to a silica membrane is dependent on ..
Charge and pH
What is the optimal pH for absorption of DNA onto a silica membrane?
7.5
3M sodium acetate can be added to a sample to ___ pH.
Lower
Name the plasmid that was used to clone the RcbL gene.
pGEM-T
What characteristic of a pGEM-T vector makes is easy to ligate PCR products onto it?
Overhangs
Name the gene on the pGEM-T vector that indicates if the RcbL gene was successfully transformed or not.
LacZ
What does the LacZ gene interact with?
B-galactosidase
The process of “introducing” ligated vectors into the E. coli is ___.
Transformation
Name the technique by which the cell walls were altered during transformation.
P-shock
The optimal temperature for “exciting the host cells” during transformation is what?
42C
After heat shocking, how was the reaction quenched?
Ice bath
Name the bacteria that was used for transformation and name one reason why it was chosen.
E. coli
Explain each of the following results: no colonies, blue colonies, and white colonies.
- No colonies -> transformation failed
- Some blue colonies, but no whites -> the ligation reaction failed
- Less than 50% white colonies -> ligation conditions suboptimal or nuclease contamination of the ligation may have occurred
- White colonies -> ligation successful
What is T4 DNA ligase used for?
- catalyzes the formation of a phosphodiester bond
- repairs single-strand nicks
- joins DNA fragments with either cohesive or blunt termini
In the protocol for isolation of plasmid DNA it states that after the resuspension, the cells should not be vortexed because of what reason?
Shearing of the DNA
Name the medium that was added to the transformed cells for the purposes of repair and recovery.
SOC medium
What temperature were the transformed cells incubated at?
65C
After isolation, DNA from three organisms was present. Name the three organisms.
E. coli, part of plasmid, rubisco
Name the two primers used to amplify the insert DNA from the plasmid.
Sp6 and T7
The fragment size for the RbcL gene should be around ___ bp.
599-750
If a blue colony was picked, the fragment size should be around ___ bp.
150
If two bands were present when the PCR product was analyzed, one at 150bp and the other at 750bp, what would they be?
- 150 would be plasmid
- 750 would be gene+plasmid
rbcL
Codes for large subunits of rubisco
GoTaq green master mix
Taq polymerase MgCl2 dNTPs Loading buffer Green loading dye
MgCl2
Acts as a cofactor to Taq polymerase reaction, acts as catalyst to reaction
dNTPs
Free nucleotides to add to growing fragment
Loading buffer
For better amplification of GC and AT rich targets
Green loading dye
Allows tracking during gel electrophoresis
Buffer AW1
Binding buffer
Buffer AW2
Wash buffer
Buffer AE
Elution buffer
Buffer AP1
Lysis buffer
Buffer P3
Neutralization buffer for plasmid preparation
PCR with primers
Two steps
- Find complimentary sequence of reverse primer
2. Rewrite 5’ to 3’ of complimentary sequence
EB buffer (elution buffer)
Elution efficiency is strongly dependent on the sale concentration and pH of the elution buffer
QG buffer (dissolve agarose and adjust pH)
Solubilizes the agarose gel slice and provides the appropriate conditions for binding of DNA to the silica membrane
PE buffer (wash filter)
Washes away salts
SOC medium
Super optimal broth is used as a recovery medium during the final stage of transformation of E. coli
Ampicillin
Prevents growth of non-transformed bacteria
IPTG
Compound is a molecular mimic of allolactose
Used to induce protein expression where the gene is under the control of the lac operator
X-Gal
To test for the presence of an enzyme
Alkaline protease
Used to inactivate endonucleases
Plant results
Cucumber, Tobacco, green bean, quinoa
