lab content Flashcards
the natural function of restriction endonucleases is in the protection of bacteria. What specific role do they play in this process?
Cleave the DNA of infecting viruses
why should you perform a very brief low speed spin in a microcentrifuge after setting up your PCR?
to collect contents at the bottom of the tubes
you have performed agarose gel electrophoresis of your amplified PCR product however you notice many unspecific bands on the gel. What could be the reason for this?
annealing temperature too low
> low annealing temperature is a common cause of nonspecific bands in PCR. When the annealing temperature is too low, the primers may bind to non-target sequences, resulting in multiple bands after electrophoresis.
how could you most accurately pipette 384micro litres?
use a p1000 to pipette 384 micro litres
in a lab report or scientific paper, where should your statistical approach be described?
methods
what could a significance value of p>0.05 suggest?
explanatory variable did not explain significant variation in our response variable
you are given a micropipette and asked to measure a volume of 500 micro litres. what is the equivalent of this in L?
0.0005L
if the pH of your Tris-EDTA (TE) buffer is 9.57, how would you adjust the pH to the desired pH of 7.5?
add HCL
you are making a solution that requires you to pipette 10 micro litres, 18 micro litres and 495 micro litres. what are the best choices of pipettes?
P10, P20, and P1000
what is the hardy-weinberg equation?
p^2 + 2pq + q^2 = 1
p TT homozygous dominant
pq Tt heterozygous
q tt homozygous recessive
after taking a buccal swab, the cheek cell preparation was heated to 55 degrees. why is this?
so that proteinase K can be most effective
what does proteinase K do?
Proteinase K is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid. Addition of Proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification.
what is EDTA?
Ethylenediaminetetraacetic acid (EDTA) is a medication used in the management and treatment of heavy metal toxicity. It is in the chelating class of drugs. This activity outlines and reviews the indications, actions, and contraindications for EDTA as a valuable agent in managing lead toxicity
The buccal swab was placed in lysis buffer that contains detergent and a chelating agent known as EDTA.
what is the role of the detergent and the chelating agent?
detergent; break cell membrane
chelating agent; stop the DNA from being broken down by DNAses
During DNA extraction, you used a mini-column assembly that is packaged with silica. what is the role of the silica in this procedure?
to trap the DNA into the column
what is the purpose of the TE buffer added at the end of DNA extraction?
to keep the DNA from degrading and store it well
what is 5ng in ug?
0.005
1ng in g?
0.000000001
The concentration of double-stranded DNA can be calculated from the equation: DNA concentration in ng/µl = A260 x 50 ng/µl x dilution factor. You add 1µl of purified DNA to 4µl of distilled water then measure the absorbance of this diluted sample at 260nm to be 0.525. What is the concentration of DNA in the stock solution?
Absorbance = 0.525
dilution factor = total volume/volume of DNA
= (4+1)/5 =5
DNAconcentrationinng/µl=A 260 ×50ng/µl×dilutionfactor
=0.525x50x5
=131.25ng/ul
ng/ul to ug/ul…. divide by 1000
= 0.131ug/ul
following a PCR reaction you obtain the following readings from the Nanodrop machine: A260=0.845;A280-0.446.
what is the A260/A280 ratio of the purified DNA?
0.845/0.446 = 1.89
is it true that provided the number of base pairs is a multiple of three, insertion of additional DNA in the coding region of a protein is unlikely to have a dramatic effect on protein function?
no, false.
could still have a significant effect on protein function
what DNA sequence might be recognised by a restriction endonuclease?
AAGCTT
discuss restriction endonucleases
also known as restriction enzymes, are enzymes that cut DNA at specific sequences, typically recognising short palindromic sequences (usually 4-8 base pairs in length).
they are widely used in molecular biology for gene cloning, DNA analysis, and genetic engineering.
recognises GAATTC (cuts between G and A
AAGCTT
GGATCC
CCCGGG
CTGCAG
GCGGCCGC
AGCT
when searching a query sequence within the NCBI database, give an example of a correct statement?
translated nucleotide query against protein database done by blastx
In a comparison of two DNA sequences found in the same location on homologous chromosomes, one of the homologues carries the sequence 5’AACTACGA-3’ and the other homologue carries the sequence 5’AACTTCGA3’.
Within a population you discover that each of these sequences is common.
What describes these sequences?
they contain a SNP that may be useful for genetic mapping
what is the number of dominant alleles in a population of 100 individuals with the following genotypes: 30BB, 60 Bb, 10bb?
BB: 30
Bb: 60
bb: 10
BB (2 B alleles)
= 30 x 2 = 60
Bb (1 B allele)
= 60 x 1 = 60
bb (0 B alleles)
= 10 x 0 = 0
=> 60 + 60 =120
what are some statements regarding single nucleotide polymorphisms (SNPs)?
they can change effectiveness of drugs between individuals
they have to occur in a significant fraction of the population (e.g. >1%)
if your A260/A280 ratio is 1.85, what does this tell you about your DNA sample?
you have a reliable DNA preparation.
this ratio is commonly used to assess the purity of DNA samples as it indicates the presence of protein or other contaminants that might affect downstream applications.
1.8-2.0 = pure DNA samples (ratios closer to 2.0 can indicate RNA contamination)
below 1.8 is protein or phenol contamination
if your DNA conc. is 0.59 ug/ul what colume would you require to obtain 25ug?
42.4ul
volume= amount needed/conc.
25/0.59= 42.37ul
