OLD Flashcards
Jeff wants to clone an insert into the polylinker pGEM-T
use enzyme that can cut at two sites to remove stuffer fragment and leave place for insert
Susan is cloning an insert into the pGEM-T vector. She chose to clone her insert into the XmnI site. Following ligation, she transformed the products in E. coli cells and plated them on medium with antibiotic and X-gal. what result?
No colonies because XmnI is in amp resistance gene
Under repressed conditions, where would LacI bind?
The lac operator
A260/280 value of 1.8 is indicative of
pure DNA
053 reading n a p10 pipette = ?
5.3 microliters
Forced cloning
digest with non complementary cohesive ends
Complementary cohesive ends?
A
- allie has a tube containing purified plasmid DNA at the concentration of 100ng/ul. She needs to add 0.5 ug to a restriction digest. What volume of her purified plasmid DNA does she need to pipette?
5 uL
- which of the following is typically found only in a CLONING vector?
lacZ gene
- what does the chemical IPTG bind to in order to induce (de-repress) expression of the GST::EGFP fusion protein in pET-41a?
lacI protein
matt is trying to clone the following piece of DNA into one of the pET-41 series of vectors in frame using EcoRI and BsrGI →what is the series of vectors called?
reading frame
if you were creating your own cloning vector, which of the following would be the best location for the multiple cloning site?
Within the lacZ gene, such that introducing an insert disrupts lacZ function
reverse primer
reverse complement of
Forward primer
compliment of sense strand?
How do you add restriction site?
add poly A tail and restriction sequence at beginning of primer
What factors should you consider when setting up the extension step of PCR protocol?
length of the target sequence
What does the forwards primer bind to?
antisense strand
in plasmid DNA purification, the procedure of anion exchange chromatography is employed to
wash out cellular impurities such as lipids and cellular proteins from the DNA prep
Natalie digested her 3 kb plasmid with PstI, the wrong restriction enzyme, which does not have any unique sites on her plasmid. She then ran the digest on an agarose gel. What would you expect to see on a gel?
multiple bands with unpredictable sizes
When performing a restriction digest what should you know about the enzyme?
- enzymes should be added last
- multiple enzymes can be used in one reaction as long as they are both active in the same buffer
- the volume of the enzyme should neve be more than 10% of total reaction
- The final concentration of the enzyme should be 1X not 10x
. cadence performed a PCR using an annealing temp of 70 C to amplify a 1 kb product. Based on Tm below, which would you expect?
annealing temp is to high-little product is formed
which of the following cannot be confirmed by performing a PCR screen using two primers that bind specifically to the vector sequence?
the orientation of the insert
- when performing a PCR screen, which of the following is true about the Tm of your primers?
they should be within a few degrees of one another
- you are attempting to perform a PCR screen to determine if your gene was cloned in the correct orientation. You expect your gene to be cloned downstream of the gst gene
reverse primer is on gene and forward primer should be on vector/gst
In which of the cases below would you need to use a specific staining method (western blot) rather than a non specific method (Coomassie)? When detecting expression of your fusion protein
when detecting expression of your fusion protein
- what is the ligand on the resin you packed your column with to purify your GST:EGFP
GLutathione
- what is the best screening method for determining whether a molecular clone has any mutations, including single nucleotide changes?
DNA sequencing
