Molecular Biology Lab Final

Question 1

What are the 3 Equations used for Concentrations?

Answer 1

(C1)(V1)=(C2)(V2)
(MW)(Vol)(M)=(g)
% = liquid/liquid or mass/liquid

Question 2

What are the steps of genomic extraction?

Answer 2

First we lyse the cells using a detergent and buffer (ATL, proteinase K, AL, Ethanol)
(ATL: degrades the tissues of the sponge) (proteinase K: degrades proteins that would interfere with PCR)(AL: promotes lysis of the cell membrane and the denaturation of proteins and DNA)(Ethanol: promotes degradation of the shell that surrounds DNA and allows it to precipitate, it also binds DNA to the matrix). The DNA is then washed using Buffer AW1 and Buffer AW2. AE Buffer releases the DNA from the matrix and elutes the DNA from the spin column.

Question 3

What are the important pieces of agarose gel electrophoresis? (How do you make it? What percentage should you use? How much voltage?)

Answer 3

• a 1% agarose gel was made in lab (75mL TAE buffer, 0.75g Agarose gel, 7.5µL EtBr)
  (1X TAE Buffer = 40mM Tris-Acetate, 1mM EDTA; EDTA chelates enzyme laces and RNA that could contaminate gel; EtBr runs the opposite direction as DNA and is excited by UV light, this dyes the samples and allows you to see them)
• Lower percentages of agarose is better for separating larger fragments
• Usually 1-5 Volts/cm is good for running a gel

Question 4

What is the ratio of loading dye used and why is it important?

Answer 4

6X loading dye was used in a 1:1 ratio which is 3X with the lowest ration possible resulting in 1X loading dye. Loading dye allows you to see the dye front as it moves down the gel and allows you to see your product when loading it into the gel, loading dye also makes the sample heavy enough to sit in the well.

Question 5

What happens in a PCR?

Answer 5

The sample is denatured, annealed, extended, and the process is repeated. It is used to amplify DNA

Question 6

What is the difference between PCR and Sanger Sequencing?

Answer 6

Sanger sequencing is like PCR but with only one of the primers used in the reaction.

Question 7

What is multiple sequence alignment and why is it used?

Answer 7

(For the purpose of DNA barcoding) Compared our sequence to known ones to deduce which species of sponge we had.

Question 8

Why is Taq Polymerase added last in the PCR Master MIx?

Answer 8

If the buffer concentration is not at the proper conditions and concentration the taq polymerase will be denatured/unfolded

Question 9

Why use Spectrophotometry?

Answer 9

To analyze the concentration of a sample.

Question 10

How BLAST work and what did we use it for?

Answer 10

Sequences are submitted to a library database, all these are cataloged. Kmer library analyzes the sequences by 25 amino acid windows. E value is expected value -> how much by chance you expect it to match another sequence. We used BLAST to identify an antigen region and select primers for that region.

Question 11

What is Expasy Prot Param and what is it used for?

Answer 11

It is used for finding molecular weight and pI

Question 12

What is BLAST used for?

Answer 12

We used BLAST to identify a suitable antigen region

Question 13

What was the purpose of the Hidden Markov Model Searches?

Answer 13

We were looking for a separable folded protein domain (a part of the sequence that corresponds to a complete independently folded domain of this protein - this makes it more likely that the antigen and proteins will naturally form the domain structure).

Question 14

What are the uses of recombinant proteins?

Answer 14

We used recombinant proteins for antibody production, but they can be used, if two are put in a tube together, to look and see if they bind together and have protein protein interactions (also good for looking at DNA protein interactions and RNA protein interactions). We can also use a gel to see if the protein is interacting with something else

Question 15

What was the choice of expression system for the second experiment done?

Answer 15

We used a bacterial expression vector with bacteria. If we were to use a bacterium to express eukaryotes it would not produce soluble protein.

Question 16

What were the plasmid properties of the Pet28a expression vector?

Answer 16

This plasmid was small and circular. The origin of replication also contained a multiple cloning site with restriction enzymes. The Pet28a plasmid also had a T7 promoter region and a lac operon.

Question 17

What are restriction enzymes?

Answer 17

Proteins with catalytic activity, they exist in bacteria to protect against viral infection.

Question 18

What were the promotors we talked about and used in lab? How does it work?

Answer 18

The promoters promote the binding and initiation of RNA polymerase to generate mRNA. The T7 promoter system of the Pet28a vectors. The T7 promoter and adjacent lac operon sequence induce to suppress uninduced expression. Also known as Bacterial Recombinant Protein Vector, it is an expression vector used for the expression of recombinant protein in E. coli.

Question 19

What are resistance cassettes?

Answer 19

antibiotic resistance of a bacteria. In this case it was Kanamycin resistance.

Question 20

How do you separate DNA from DNA?

Answer 20

In terms of bacterial genomic DNA and plasmid DNA, we exploited the size difference between the bacterial genome and plasmid DNA, even though they are both double stranded. Through the treatment with an alkaline lysis buffer that denatures the DNA, the small pieces are able to re-complement and the large pieces just turn into a tangle.

Question 21

How does column purification work?

Answer 21

The DNA binds to the column, ethanol removes the water shell from the DNA, the DNA is washed, and then it is eluted from the column and water is added back to it.

Question 22

What is transformation?

Answer 22

It consists of inserting a foreign plasmid or ligation product into bacteria.

Question 23

What happened in the small scale pilot expression?

Answer 23

By using pre-induction and post-induction (by IPTG to induce protein production for purification) prepared samples, we were able to see if the induction and expression worked so we could proceed with the full scale induction.

Question 24

What was the purpose of the full scale expression?

Answer 24

In the full scale expression the sample’s cells were lysed and collected to isolate the His6-GFP protein from Sidc and purify it. The fractions were used to find out if the protein was in the soluble or insoluble sample and the elution sample was used to see if the protein was purified and isolated.

Question 25

How does T444T function to produce RNA and how does it differ from Pet28a?

Answer 25

T444T has two T7 sites that create a sense and antisense strand of complementary RNA to make a double stranded RNA. Pet28a only had 1 T7 promoter.

Question 26

How does the TA cloning strategy work?

Answer 26

It is a cloning technique. In TA cloning the plasmid and gene are ligated together, transformed into bacteria, checked to make sure the desired fragment is there, the DNA is digested and fed to E. coli.

Question 27

What enzymes did we use and why?

Question 28

How doe self-ligation work?

Answer 28

Plasmid prep of T444T is done to make it empty and circular, it is run on a gel, we then found we had multiple different species of the plasmids. Then we did an EcoRV digest and ended up with the right sized band for the conformation of plasmid that we wanted (linear plasmid). We then purified the plasmid, did T-tailing, and rand a ligation so the plasmids without Ts would close up on themselves. This was run on a gel and then the linear plasmids were cut out of the gel and purified. Then the T-tailed plasmids were ready for ligation with the A-tailed plasmids.