Freshmen Waksman Notes Ch. 1

Name the 2 types of libraries

Genomic and cDNA

What is a library?

Random collection of DNA fragments. Ideally contains one or more copy of every DNA sequence in an organism.

Use of libraries:

Isolate specific DNA fragments of interest

Simple analogy of a vector is a:

car beep beep

Vector type in waksman:

Plasmid

Plasmid definition:

Circular molecule of DNA found in bacteria, archea and yeast.

What does the origin of replication do on the plasmid control?

_______ controls host range (where _____ can exist) and copy number (rate of replication).

Plasmid Vector we use in Waksman:

pTripleEx2

Resistances on pTripleEx2:

Ampicillin (an anti-biotic, found in agar) resistance and LacZ gene.

Where does our insert go in pTripleEx2 ?

Between the LacZ gene

What is step 1 in amplifying?

Digest: Cutting our insert and vector with restriction enzymes.

What is step 2 in amplifying?

Ligate: Paste/connect insert into pTripleEx2 at the SFiIA and SFiIB sites.

What is step 3 in amyplifying?

Introduce the plasmid into bacteria colonies after treating cells with CaCl (makes cell membrane more positive)

Plasmid multiplies inside cell, then cell multiplies.

Yep, this shows that the insert multiplies independently of the host' genome.

How do we know bacteria has inserts?

With our selectable markers (ampicillin resistance and LacZ gene)

What does the ampicillin resistance do?

Since our agar plate has ampicillin, only bacteria with the plasmid can grow.

What does LacZ do?

LacZ gene produces beta-galactosidase which cleaves enzyme substrate X-Gal. X-Gal gives colony blue color. Cleaving it makes our colony white, meaning plasmid with insert is in the bacteria colony.

What and where is the multiple cloning site?

The multiple cloning site is on the plasmid between the LacZ gene. Allows us to clone our insert into LacZ gene. Insert is between SFIiA and SFIiB sites.

What is a genome?

All of the genetic material from an organism (coding and non-coding)

Genomic library definition:

A collection of randomly generated DNA fragments from an organisms genome.

What does the "c" in cDNA library stand for?

cDNA = ComplementaryDNA. It is complementary to RNA.

In a cDNA library you make a complementary DNA strand of the mRNA. Why not use/make a copy of the DNA the mRNA came from?

Because the mRNA is processed RNA transcript, it is made to make protiens. This means the mRNA has no introns (non coding regions) and will only be the coding genes. DNA has both introns and exons

Regarding cDNA libraries: Why do you need to make a complementary DNA strand to the mRNA?

mRNA is single-stranded and unstable meaning it can't be stored in the lab for a long time.

Biggest difference between genomic and cDNA libraries: (in-depthish)

Genomic libraries aren't converted into mRNA then back into DNA. Because it lacks these steps it will contain the whole genome including the non-coding regions. The cDNA will contain only the coding regions and expressed genes which is what we want.

What do you use to separate the mRNA from other RNAs during the second step of constructing a cDNA library: Purify mRNA?

Oligo dT column will bind to the poly-a tail of the mRNA because it is a string of Ts and the other RNAs will get washed through.

How many; And what are the steps of constructing a cDNA library?

7 steps. Simply:

1. Isolate mRNA

2. Purify mRNA with Oligo dT

3. Reverse transcribe to make a DNA:RNA hybrid

4. Remove RNA from DNA:RNA hybrid

5. Double strand synthesis (replication) DNA:DNA

6. Ligate double-stranded cDNA into pTripleEx2

7. Transform pTripleEx2 into bacteria

What age is the duckweed we are getting mRNA from?

Mixed age to maximize diversity because not all genes are expressed at once.

What method do we use to remove the RNA from the DNA during step 4 of constructing a cDNA library: Remove RNA from DNA:RNA hybrid?

Hydrolyze with a strong base like NaOH to remove the mRNA.

What is the enzyme responsible for step 3 of constructing a cDNA library: Reverse transcribe to make a DNA:RNA hybrid?

Enzyme Reverse Transcriptase (derived from retrovirus)

During step 6 of constructing a cDNA library: ligate into pTripleEx2, what are the restriction sites on the plasmid called?

SFiIA and SFiIB