exam 1 Flashcards
What is the ideal primer length? What is the limit?
Ideal 18-24bp, can go up to 42
What is the ideal GC content of primers? What if it is not in those bounds?
Ideal 40-60%. Increase means higher temp for denature and secondary primer structures wont denature, lower means less stability
What is the difference of annealing temperature and melting temperature?
2-3C lower
What occurs during the melting temperature for primers?
Temperature where 1/2 old the DNA duplex dissociates and becomes single stranded
What is the ideal melting temperate range?
52-65C
What is the concern of primer secondary structures?
Can lead to poor or no yield of product/application fails
What are the common primer secondary structures?
Hairpins, self-dimer primer, heterodimer
What is the max number of repeats in primers?
4
What is deltaG? What are its limits?
-10 to +10. Indicates the probability of primer secondary structures forming
What are the three steps of PCR?
Denaturation, annealing, elongation
What does PCR stand for?
Polymerase chain reaction
What occurs during Denaturation? What is its ideal temperature?
94 or 95C. H bonds between complimentary bases are broken. Double to single stranded
What occurs during annealing? What is the ideal temperature?
Primers bind to their complimentary sequences. Occurs at 55C (52-64avg)or 2-3C lower than Tm
What is another term for annealing?
Primer binding
Do the primers become a part of the new PCR product? T/F
True!
What are the two types of primers?
Forward and reverse complementary
What occurs during the elongation step? At what temperature?
DNA polymerase binds to primers and adds dNTPS(new bases) to finish the new strand. Occurs at 72C
What is another term for elongation?
Polymerization
How is the elongation step controlled?
Time. Length of the step controls the length of complimentary sequence
In what direction does the elongation step occur?
5’ to 3’
What is another term for primers?
Oligo nucleotides/ oligos
What is the primer directionality? Where does it bind?
Bonds to the 3’ end of the template strand and is in the 5’ to 3’ direction
Can forward and reverse primers be different lengths?
Yes
Within what range do the annealing temperatures of primers need to be to ensure they both bind?
Within 1C
How is the forward primer determined?
Right from the start of the sequence, same as the 5’ to 3’ template from the start
How is the reverse primer formed?
Compliment of the 5’ to 3’ template on the 3’ end and then “flip it” or find the reverse compliment
Do you compliment the forward primer?
NO, only the reverse
What is a restriction enzyme? What does it do?
An enzyme that functions in recognition and cutting of DNA sequences
Will a restriction enzyme cut DNA if the recognition sequence is changed?
No
What does the restriction enzyme bind to?
Double stranded DNA
Where do restriction enzymes cut?
Phosphate backbone of DNA
How do you determine the concentration of DNA in a sample?
Multiply the OD 260 by 50ug DNA/mL
What is the ratio that determines a pure DNA sample? What if it is outside of that range?
A pure sample is 1.8-2.0. Outside of this range it is contaminated by protein
What causes a nonspecific band?
When primers bind to the non target region and make smaller copies of different spots in the sequence
Is ladder separation linear? T/F
True, they move by size
How do DNA fragment sizes move in gel?
Larger fragments move slower and closer towards black
Smaller fragments move faster and further towards red
How long are restriction endonuclease typically?
4,5,6,8bp or Bivalent but it is dependent on the target length of the sequence
How do you write SNPs?
1st possible nucleotide- spot in sequence - 2nd possible nucleotide
C 145 G
How many locations differentiate the locations in the gene of a taster vs nontaster?
3
What does SNP stand for? What does it mean?
Single nucleotide polymorphisms, where nucleotides in the sequence differentiate
What is Chelex? What if it gets into your PCR sample?
Chelex are nano-beads of Mg2+ and Ca2+ that remove metal ions and will inactivate nucleases that will break down DNA. If it gets into your PCR then it inactivates Taq polymerase since its cofactor is Mg
What is anthocyanin in plant cross? What gene is this? What expression is dominant?
Purple pigment, ANL gene, ANL is dominant over anl
What is the ROS gene? What does it indicate? Which expression is dominant?
Rosette dwarf gene which gives tall or short plant. Dominant expression is ros/ros
What are true breeding parents?
Genotypes of homozygous dominant or recessive and we will see the phenotype in all generations. Ex Homozygous dominant ROS/ROS will give tall offspring always
What do “fluffy clouds” mean at the bottom of gel electrophoresis runs?
Primer dimers
What does the percentage of a gel mean in gel electrophoresis?
A higher percentage gel means resolving optimal resolution of the linear DNA size you can see
What is the purpose of loading dye?
To add to DNA samples for PCR to make it sink to the bottom of the well to not affect migration in the buffer and not the gel
What is agarose gel composed of?
A buffer/salt solution of polymers that sort out DNA fragments by size
What is the purpose of agarose gel electrophoresis?
To separate DNA fragments by size
What is the purpose of the gel solution (2)?
Maintains pH and salt concentration
Why does DNA move towards the positive (red) electrode (2)?
It is negatively charged at a neutral pH
What happens when you increase the agarose concentration?
Pores become smaller
What type of relationship is there between DNA size and migration?
Logarithmic relationship between the DNA fragment weights and distance traveled
What is the purpose of having dye in the gel itself?
To visualize DNA and ladder under UV light
What concept do we use when calculating the amount of loading dye?
Equivalent volumes
Do equivalent volumes equate to 1:1? T/F
False!
What equation do we use to calculate the amount of loading dye for a reaction?
Ve/(n1-n2)
Ve= volume of rxn
N1=initial dye concentration (typically 6x)
N2= final dye concentration (1x)
How do we make a cocktail reaction for PCR?
Total amount of reactions plus 1 for pipetting error
What is typically included in a PCR master mix (4)?
dNTPs, Taq, buffer, Mg ions
What does “diluted to” mean?
The initial volume of solute is diluted to the total volume of solute + diluent
1mL phage is diluted to 4mL =1mL phage +3mL diluent
What does “added to” mean?
Refers to volume of diluent
1mL phage is added to 4mL broth. 1/5 dilution
What is the equation for dilution?
D= Vsolute/(Vsolute+Vdiluent)
What are the two types of percent solutions?
Volume/volume or weight/volume
Why do we avoid more than 3 G or C in the last 5bp at the 3’ end of the primer?
Avoids primer secondary structure
How to go from L to mL? mL to L? L to uL?
L to mL *1000
mL to L /1000
L to uL *10^6
How to go from M to mM? mM to M? M to uM?
M to mM *1000
mM to M /1000
M to uM *10^6
What units are used in weight/volume percent solutions?
Gr/mL
What units are behind molarity?
Moles/L
