Molecular Biotechniques Quiz 2

What is reverse transcription?

Reverse Transcription is the process of transcribing RNA to generate DNA (in this case, cDNA)

What is needed in a reverse transcription experiment?

• Template: RNA

• Primer: oligo dTs (string of thymines), random hexamers, gene specific

• RNA-dependent DNA polymerase (aka reverse transcriptase)

• Nucleotides (added to primer by RT)

What are the reactions (experimental and controls) that were set up in reverse transcription?

+RT, -RT, and -RNA

Why were each of these reactions run?

• +RT is the positive control which has all the reaction components

• -RT is the negative control containing no reverse transcriptase. Since no RT is in the reaction, no cDNA can be made. Therefore, this control shows that any product in the PCR sample is due to gDNA contamination.

• -RNA is the negative control that has no RNA, which therefore means there is nothing to provide a template. Any product in this PCR sample is due to contamination of one of the reaction components.

How long do primers need to be to be specific?

18-24 nucleotides in length

How many G+C’s should be in a primer?

40-60% GC content (should have similar amounts of C+Gs and A+Ts)

How do you write the sequence of forward and reverse primers?

All sequences are from 5’ to 3’

• Forward primers: taken directly from the sequence. Eg, if given ACGTA, the primer would be written 5’-ACGTA-3’

• Reverse primers: take the reverse complement of the sequence of the other end of the given sequence. Eg, if given ACGTA, the reverse would be ATGCA, and the complement would be TACGA, which would then be written 5’-ATGCA-3’

What do primers do in a PCR sample?

2 Primers (Forward and Reverse)

• Forward Primer: Binds to the 3' end of the template strand. It allows the DNA polymerase to start adding nucleotides in the 5' to 3' direction, creating a complementary strand moving toward the target region

Reverse primer

• Reverse Primer: Binds to the 3' end of the complementary strand. It enables the polymerase to build the complementary strand in the opposite direction, toward the forward primer.

Why is there template in the PCR sample reaction?

The template contains the actin DNA sequence. The template DNA in PCR provides the specific sequence that primers bind to, allowing DNA polymerase to copy it. Without the template, there’s nothing to amplify in the reaction.

What is DNA polymerase used for in PCR sample?

DNA polymerase adds nucleotides to make complementary strand of the original strand

What are dNTPs used for in PCR sample?

dNTPs add nucleotides to the other (unzipped) strand to the 3' end of DNA during extension step

What are the three steps in a PCR cycle? What is their order and what happens in each step?

1. Denaturing: 90-95C → bonds are broken from high heat and strands separate

2. Annealing: 45-65C → temp is lowered for primers bind to complementary template

3. Elongate: 72C → DNA polymerase adds nucleotides to synthesize new strand

What is TOPO?

TOPO is an enzyme called topoisomerase, which can cut and rejoin DNA strands. In cloning, it’s used to insert DNA fragments quickly and efficiently without the need for DNA ligase.

What is TOPO attached to and what does it do in the cloning reaction?

TOPO is covalently attached to the ends of a linearized cloning vector (plasmid). It facilitates the insertion of the PCR pro

What is meant by “TA”?

“TA” refers to the overhangs on DNA fragments where a single thymine (T) is added to the 3' ends of the vector, and the PCR product has a complementary adenine (A) overhang. This allows the PCR product to easily ligate into the vector through complementary base pairing

Does TA work with all PCR products made by all polymerases?

No, it mainly works with PCR products made by Taq polymerase, which naturally adds a single A overhang to the 3' ends. High-fidelity polymerases (like Pfu or Phusion) often leave blunt ends, so they typically require a different cloning strategy.

Transformation is the introduction of DNA into cells. A few times this semester we’ll be putting plasmid DNA into competent cells – what are competent cells?

Competent cells = cells that can take up DNA

Are all cells competent cells, all of the time?

Cells are not competent cells all the time – you need to make them competent via chemically or electrically

Once we have added DNA to competent cells, we need to have a way to select for cells that have taken up DNA – usually this is done by growing cells on plates or in media that has antibiotics added to it. Only cells that have taken up the plasmid will be able to survive … why?

Because the cells with the plasmid contain the antibiotic resistant gene which allows them to grow on the ampicillin plates

For our TOPO-TA cloning, we saw that there was another selection/screening method. What is blue/white screening, and what does it tell you about the colonies on the plate?

Blue-white screening is a selection method that differentiates between white colonies and blue colonies.

White colonies have a nonfunctional beta galactosidase (or in other words, a beta galactosidase that does not have its alpha subunit) and therefore can not cut X-gal.

Blue colonies contain beta galactosidase with its alpha subunit and therefore are able to cut X-gal.

The purpose of using blue-white screening is to see which plasmids have taken up the insert (the white colonies) and which ones do NOT have the insert (the blue colonies).

What could the inserts on white colonies possibly contain?

• The desired actin gene

• Primers

• Nucleotides
  Any DNA that was in RT PCR