What are the commonly used pipettes in the labs and their range of use? What is
ID: 202354 • Letter: W
Question
What are the commonly used pipettes in the labs and their range of use?
What is accuracy of an instrument? How is it determined
What is precision of an instrument? How is it determined
Acceptable limit for the error in accuracy?
Acceptable limit for the average deviation in measuring precision?
Optical density of bacteria in suspension? What does it measure? Any units
How do you transfer from one tube to another, how do you hold the tubes
How do you transfer bacteria into a Petri dish, how do you hold the Petri dish?
How do you streak a plate to isolate pure colonies of bacteria?
Why do you need to streak bacteria on a plate?
How do you incubate your liquid bacterial cultures? And why?
How do you incubate your Petri plate cultures? And why?
What do you use the filter paper discs for in testing for efficiency of disinfectants?
Genomic DNA vs. plasmid DNA?
pBluescript SK-?
Features of vectors?
Plasmids Extra-chromosomal DNA?
Features of plasmids Selectable Markers Bla: Beta lactamase?
Alkaline lysis method for plasmid isolation?
Qiagen system kit for plasmid DNA isolation?
Description of the procedure for the Alkaline Lysis and why do we do each step?
Which electric direction the DNA runs in gel electrophoresis?
Why do we have to add a loading buffer with the DNA sample in the agarose gel?
DNA migrates in the agarose gel according to its size and shape, which molecules migrate faster? smaller or larger?
Why do you load a DNA marker on the gel?
What are endonuclease enzymes?
The two types of restriction endonuclease enzymes: Symmetric and Asymmetric cutters?
What are DNA cohesive ends (also called sticky ends)?
Why do the multiple cloning sites have unique RE sites?
Linearization of plasmid DNA (how and why)?
The DNA insert: 1.3 KB fragment, which enzyme is it cut with and why?
What does the insert that used in this lab code for?
Why did we use Shrimp Alkaline Phosphatase?
Why did we heat the Shrimp Alkaline Phosphatase to 65 C?
DNA Ligase joins the _________(which end 5’ or 3’?) end of one nucleotide to the --
---- (5’ or 3’ end) of another nucleotide.
What are blunt ends of the plasmid DNA or fragment DNA?
Why do we cut the plasmid and the fragment with the same restriction enzyme?
What factors do we have to consider in choosing the restriction enzyme to cut the
plasmid and the fragment?
What are the factors that we consider in mixing the plasmid and the insert (fragment)
in setting up the ligation reaction mix?
What is the purpose of ligase enzyme?
What is bacterial transformation?
What is positive selection
What is negative selection
alpha complementation (Blue White color selection):
What are competent bacterial cells
What enzyme does Lac Z code for? (-galactosidase)
What does the Lac Z gene part on the plasmid code for?
What does the Lac Z gene part on the bacterial chromosome code for?
What color colonies are produced by bacteria that did not take up any plasmid, and
grown on media containing Amp/X-gal/IPTG?
What color colonies are produced when the non-recombinant plasmid is taken up by
the bacteria and subsequently grown on LB/AMP/X-gal/IPTG plates?
What color colonies are produced when the recombinant plasmid pSKII-/Kan was
taken up by bacteria and subsequently grown on LB/Amp/X-gal/IPTG plates?
How do you confirm that your colonies have your fragment insert (the gene of
interest)? What are the steps that you will do starting from detecting the colony
growth on your plate?
Which restriction enzymes you used to analyze the recombinants and what is the
purpose of each one of them?
What is the method of isolation of DNA that you will use
What is the end product
What are the enzymes that you will use in this lab
Review the ingredients of the solutions in the DNA plasmid isolation protocol
(Alkaline Lysis) in labs 3 and 6, their purpose and their function of each step.
What is Ethidium Bromide, how does it stain DNA?
What size is the DNA insert we used in this lab? What did the insert code for?
What size is the resulting recombinant plasmid DNA (pBlueKan)?
On which media (+antibiotics) the bacteria containing the recombinant DNA plasmid
will grow?
Transformation protocol (why’s) and transformation efficiency equation.
Competent cells
Why does the insert go in the plasmid in either orientation?
Why did we pick bacteria with potential recombinant DNA that has the gene in either
orientations
Explanation / Answer
You have asked too many questions in this. That is not as per chegg policy. But will give the short answers of these questions.
What are the commonly used pipettes in the labs and their range of use?
Ans: Generally 1ml pipette( 1000ul- 100ul), 200ul pipette( 200ul-10 ul), 20 ul pipette ( 20ul -2ul) and 10ul pipette ( 10ul-0.2ul).
What is accuracy of an instrument? How is it determined
Ans : accuracy is determined by the experimental value is how close to theoretical value.
What is precision of an instrument? How is it determined
Ans: How many time a value repeated or come near to the same value that any instrument measures. It is generally determined by statistical method that is stand deviation.
Acceptable limit for the error in accuracy?
Acceptable limit for the average deviation in measuring precision?
Optical density of bacteria in suspension? What does it measure? Any units
Ans:Optical density of bacterial measures at 600 nm. There is no as such unit for this. This is measurement of scattering in the path of light.
How do you transfer from one tube to another, how do you hold the tubes
Ans: In this process one tube is slightly tilted so that material transfers through the wall of the tube. This method adopts to avoid the frothing.
How do you transfer bacteria into a Petri dish, how do you hold the Petri dish?
Ans : petri plate should be tilted and loop should near to 45 degree so that it will avoid andy damage to agar surface. During streaking concentration of bacteria will reduce and finally get the single isolated colony.
How do you streak a plate to isolate pure colonies of bacteria?
Ans : there are several techniques of streaking but sector wise is streaking is best that gives the single isolated colony.
Why do you need to streak bacteria on a plate?
Ans : so that whatever culture is obtained is pure culture or orginate from single bacteria. Koch’s postulates.
How do you incubate your liquid bacterial cultures? And why?
Ans : Inoculum should be from single colony and it should be from single isolated colony. This islolated colony should transfer into liquid media with the help of sterile loop.
How do you incubate your Petri plate cultures? And why?
Ans: it depend upon the given cultural conditions, temperature and aerobic and aerobic conditions. Generally plates kept inverted in incubator in order to avoid the moisture contact with grown bacteria.
What do you use the filter paper discs for in testing for efficiency of disinfectants?
Genomic DNA vs. plasmid DNA?
Genomic DNA is big and non circular in nature whereas plasmid DNA is reltively small and circular in nature.
pBluescript SK-?
Is a plasmid that used for the cloning and here SK represents the orientation of Sac I site that is near to Lac Z gene. And here (-) is represent the orientation of f1 intergenic region.
Features of vectors?
Plasmids Extra-chromosomal DNA?
Yes, that replicates independently.
Features of plasmids Selectable Markers Bla: Beta lactamase?
Ans : this gene encodes for enzyme beta-lactamase that degrade the ampicilline antibiotic .
Alkaline lysis method for plasmid isolation?
Ans : alskaline medium with SDS entrap all the protein and also denature the DNA. Here genomic DNA will not re anneal due to their size but plasmid will re anneal due to its small size.
Qiagen system kit for plasmid DNA isolation?
Description of the procedure for the Alkaline Lysis and why do we do each step?
Which electric direction the DNA runs in gel electrophoresis?
Ans: Toward the positive electrode because DNA contains negative charge.
Why do we have to add a loading buffer with the DNA sample in the agarose gel?
Ans: It contains glycerol that keep the DNA sample into well and dyes that show the movement of DNA.
DNA migrates in the agarose gel according to its size and shape, which molecules migrate faster? smaller or larger?
Ans: If all the plasmid are same size than super -coiled will move faster as compare nicked and linear form.
Why do you load a DNA marker on the gel?
Ans : it will give the estimation of molecular weight size of our DNA.