Primer Puzzles
After digestion and then ligation into a plasmid digested with the same restriction enzymes, restriction sites engineered onto primers (and ultimately becoming part of the amplicon) are:
a. LOST
b. RE-CREATED
c. ONLY HALF-SITES REMAIN
B
The restriction sites are re-created when the single-stranded sticky ends H-bond, and you add ligase or topoisomerase to covalently close the DNA with a phosphodiester bond.
A gradient PCR thermal cycler changes the temperature across the sample bed from left to right. This thermal cycler would allow you to optimize which aspect of your PCR?
Annealing temperature
You know that your plasmid is 3000 bp. What number/conversion factor must you know to convert it to find out its MW?
1 bp = 646 g/mole
So 3000 bp x 646 g/mole/bp = 1.938 x 106 g/mole
You need a 10:1 insert:vector molar ratio for a blunt-ended ligation. Your vector is 3000 bp and your insert is 300 bp. Both are at a concentration of 200 ng/uL. How much do you add of each?
a. equal amounts
b. more vector
c. more insert
a. equal amounts
Your insert is 10X smaller than your vector, so there are 10X more molecules per (unit mass) than of your vector at an equivalent mass.
Which weighs more, a mole of a 10 kb piece of DNA, or a mole of a 1 kb piece of DNA?
The 10 kb piece
10,000 bp x 646 g/mole/bp x 1 mole = 6,460,000 g
1000 bp x 646 g/mole/bp x 1 mole = 646,000 g
Your primers are predicted to form a heterodimer, with a 5' overhang. The delta G is -3. Is this problematic?
Yes.
Taq can extend the 3' OH end, making the dimerized region much longer and making the delta G much more negative in real life, IF you allow primer dimers to form.
There is too much Mg++ in your PCR, your primers are random hexamers, and your template is chromosomal DNA. What is the probability of mispriming?
a. High
b. Neutral
c. Low
A. High
You have short, nonspecific primers, and a very long template where they are likely to bind >> once. You stabilized any mispriming with too much Mg++ . You're basically hosed!
How much does one mole of a 10-nucleotide primer weigh? (Note: nucleotides, not basepairs)
3230 g
The MW of 1 bp is ~646 g/mole in dsDNA, so the MW of 1 nt is 323 g/mole. 10 bp is 3230 g/mole.
You need a 3:1 insert:vector molar ratio for a sticky-ended ligation. Your vector is 3000 bp and your insert is 1000 bp. Both are at a concentration of 200 ng/uL. How much do you add of each?
a. equal amounts
b. more vector
c. more insert
a. equal amounts
Your insert is 3X smaller than your vector, so there are 3X more molecules per (unit mass) than of your vector at an equivalent mass.
A multiple cloning site of an plasmid is cut with SmaI, a blunt-cutter. In a ligation with an insert also cut with SmaI, what are possible ligation products?
a. Vector ligated back together
b. Two or more vectors ligated together
c. Two or more inserts ligated together.
d. Vector and insert ligated together.
e. All of the above.
e. all of the above
What PCR product(s) would form if you accidentally added only reverse primers to a PCR?
Anchored products only, and only from the 5' end of the reverse primer, not from the other end of the target region.
Which amino acids in the catalytic site of Taq require a divalent cation to stabilize the transition state?
Aspartates
109 copies of template DNA is:
a. greater than one mole, or > 6.02 x 1023 molecules
b. less than one mole, or < 6.02 x 1023 molecules
less than one mole, or < 6.02 x 1023 molecules
You need a 3:1 insert:vector molar ratio for a sticky-ended ligation. Your vector is 3000 bp and your insert is 3000 bp. Both are at a concentration of 200 ng/uL. How much do you add of each?
a. equal amounts
b. more vector
c. more insert
c. more insert
Your vector and insert have the same number of molecules for an equivalent weight, so add 3X more insert.
A multiple cloning site of a plasmid is cut with SmaI, a blunt-cutter. In a ligation with an insert also cut with SmaI, what are possible ligation products that will allow survival of E. coli if they are transformed into competent cells, and plated on LB+amp? Choose all that apply.
a. Vector ligated back together
b. Two or more vectors ligated together
c. Two or more inserts ligated together.
d. Vector and insert ligated together.
e. All of the above.
A, B, and D.
The vector always has the selectable marker and the origin of replication, necessary for maintaining the plasmid in E. coli.
What would you see on a gel, if you accidentally added only forward primer to your PCR?
Nothing.
The PCR would only make anchored products on one side, but no amplicons, and there would be no exponential increase in dsDNA so not enough dsDNA for EtBr to bind and become visible.
Future you is mixing up dNTPs for your lab to use in PCRs. You add CTP, GTP, and TTP, but forget to add ATP. What will happen to the first PCR someone does, whose primers and target are below?
5' TCT CCG ACG TAT
3' AGA GGC TGC ATA TAC CCA AAT GCG CCT TAA...
Nothing!
Taq will stall at the first nucleotide, having nothing to add.
You want one nanomole of each primer (16 bp) and one nanomole of template (1600 bp). What is the ratio of weights of each primer/template?
1:99 (the fraction is 1/100)
16 bp x 646 ng/nmole/bp x 1 nmole = 10,336 ng
1600 bp x 646 ng/nmole/bp x 1 nmole = 1,033,600 ng
10,336 ng/1,033,600 = 1/100.
You need a 3:1 insert:vector molar ratio for a sticky-ended ligation. Your vector is 3000 bp and your insert is 3000 bp. Both are at a concentration of 200 ng/uL. How many ng of each do you add, for a total of 400 ng DNA in the ligation?
300 ng insert, 100 ng vector
Need 3X more molecules of insert, and they are equivalent length, so add 3X more insert mass
The pCR4-TOPO vector was made by digesting with a blunt-cutting restriction enzyme, and synthetically adding thymidines to the 3' ends. Which can this vector do in a ligation?
a. self-ligate
b. ligate to an insert with As added to the 3' ends
c. ligate to an insert with As added to the 5' ends
d. none of the above
c. ligate to an insert with As added to the 5' ends
You fuse an ORF of interest to the 3' terminus of the GFP ORF to see its location in tissues. Should you include the stop codon when you amplify your ORF?
Yes.
The 3' end of the GFP ORF corresponds to the C-terminus of protein. You need the ribosome to stop translating there.
If 10 pg of a 3 kb vector = 2.85 x 106 molecules, then how many molecules are in 10 pg of a 6 kb vector?
a. 2.85 x 103 molecules
b. 1.425 x 106 molecules
c. 5.7 x 106 molecules
d. 5.7 x 1012 molecules
b. 1.425 x 106 molecules
You need a 1000:1 primer:template molar ratio. Your primers are 6 bp and your template is 6000 bp. Both are at a concentration of 200 ng/uL. How much do you add of each?
a. equal amounts
b. more primer
c. more template
a. equal amounts
Your primers are 1000X smaller, so there are 1000X more molecules of them for an equivalent weight of template.
You need a 3:1 insert:vector molar ratio for a sticky-ended ligation. Your vector is 3000 bp and your insert is 3000 bp. State algebraically how you'd determine how much mass to add of each, for a total of 400 ng DNA in the ligation?
1X + 3X = 400 ng
(3 + 1) X = 400
4X = 400
X = 100
X = vector; 3X = insert
Instead of pure insert, you add a shot of a completed PCR reaction to pCR4-TOPO, and allow ligation. Your PCR template had an ampicillin resistance gene, and so does pCR4-TOPO. If you transform your ligation mix into competent E. coli, which might you harvest from a plasmid prep of the putative transformants? There might be more than one answer.
a. pCR4-TOPO::insert
b. pCR4-TOPO::pCR4-TOPO
c. pCR4-TOPO::your template
d. your template
e. your insert
A and D