DNA and Plasmids
PCR and gel electrophoresis
Cloning and transformation
Protein and enzymology
Lab techniques and safety
100

What are plasmids?

Small circular DNA molecules used in cloning.

100

What is Taq polymerase?

This enzyme copies DNA during PCR.

100

What LIC stands for?

Ligation Independent Cloning

100

What is the enzyme that hydrolyzes starch?

Amylase (alpha amylase).
100

What is a thermocycler?

This equipment heats and cools samples during PCR.

200

What is a restriction enzyme?

An endonuclease used to linearize the plasmid.

200

What is the melting temperature (Tm) of the sequence below?

AGGTCTAGGCTTAAGCG

A/T - 8

C/G - 9

Tm= (A/T x 2) + (C/G x 4)

Tm= 8x2 +9x4

Tm=52 0C

200

Why we have to transform BL21 cells?

Because the have the T7 RNA polymerase.

200

What is starch?

Is a plant-based polymer of glucose.

200

True or false: I should wear gloves all the time during a lab class.

TRUE!

300

Why do we have to do a plasmid clean-up after digesting the plasmid?

To remove the restriction enzyme from the solution.

300

What is the annealing temperature of a primer that the Tm is 63 degrees Celsius?

58 degrees Celsius.

300

Why E. coli cells carrying the sacB gene don't grow in the presence of sucrose?

Because they convert sucrose into levansucrose, which is toxic to the cells.

300

What is the metal molecule most used to bind to the his-tag?

Nickel.

300

What is a critical step while loading a centrifuge with tubes?

Balancing the centrifuge placing tubes across from each other or adding a tube with same volume of liquid if the number of tubes is odd.

400

I am using the plasmid p15TV-L to clone the mcbA gene. After transforming the DH5a cells, I could only see grown in the plate without sucrose. Why?

Because the sacB gene was not removed.

400
What is the purpose of a colony PCR?

To confirm the gene insertion in the right location.

400

What to do if we want to have a gene inserted into a plasmid in only one possible orientation?

Use 2 different restriction enzymes.

400

Why do we have to add IPTG to the LB media when growing the BL21 cells?

To remove the Lac operator inhibitor from the Lac operator and allow the T7 RNA polymerase to work.

400

You accidentally vortex your plasmid sample too hard after miniprep. What might happen?

Shearing or degradation of the plasmid DNA.

500

I am digesting a plasmid with 2 restriction enzymes. One of them has 2 binding sites in the plasmid. How many band in an agarose gel will I see if I run my sample in a gel after digesting the plasmid?

3 bands.

500

Design the forward and reverse primers for the following sequence? 18 nucleotides long each.

GCGGCTGCGAGTGCTGAAACGGCGAACAAATCGAATGAGCTTACAGCACCGTCGATCAAAAGCGGAACCATTCT

Fw: GCGGCTGCGAGTGCTGAA

Rv: AGAATGGTTCCGCTTTTG

500

What does it mean if the DH5a cells only grow without sucrose?

It means that the sacB gene was not removed from the plasmid.

500

Why not using lactose instead of IPTG to induce protein expression?

Because IPTG is not metabolized by the cells.

500

A student’s DNA sample shows an A260/A280 ratio of 1.2. What does this indicate?

Indicates that there is too much protein contamination in the DNA solution