Experiment 2 - Isolation, cloning and transformation of plasmid DNA

1. chromosome or
2. extra chromosomal piece of DNA ie\ phage or plasmid
antibotic resistance in bacteria may be carried on what
1 *cloning, sequencing and expressing* foreign
genes in E. coli

1c. A DNA squence with MCSintroduced into this region

1. pUC useful for

1c. elaborate on the lacZ'(alpha-complementing) region of each plasmid

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1. isolate plasmid DNA – pKan and pUC18

1a. The pUC plasmids contain:
– [pBR322] ampicillin resistance gene [the beta-lactamase (bla) gene] – origin of DNA replication

1b. plasmid pKan contains a kanamycin resistance gene.

2. When introduced into a suitable E. coli DH5a host strain carrying a lacZ portion that is able to
complement the portion on the plasmid, plasmid ligate to that portion of the lacZ gene as well as MCS –> transformed strain

X-Gal [(5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside)] is an inert chromogeni substrate –> cells with LacZ are able to make beta-galactosidase –> hydrolyzes X-Gal on plates into [colorless galactose and 4- chloro-3-brom-indigo, forming an] intense blue precipitate.

4. Cloningligating restriction DNA fragments (kanamycin resistance gene) into the Multiple Cloning Site of pUC18 vector = recombinant plasmid –> inactivates the lacZ gene, therefore cells of E.coli strain DH5a transformed with these new recombinant plasmids can not make betagalactosidase on selective media (x-gal)
and therefore produce white colonies on the agar plates –> indicate the presence of the kanamycin gene inserted into the pUC18 vector.

6. These colonies will then be streaked onto kanamycin/ampicillin (or carbenicillin) plates
to verify the presence of the kan and amp gene in the vector. E.coli DH5a colonies with resistant to both will be selected

1. Isolate what
1a. puc plasmid contains what
1b. pkan plasmid contains what

2. Describe the host
what does the plasmid do
what is x-gal
molecular process behind blue colonies (hint substrate on plate)

4. what to do with pkan plasmid and its effect

6. final step

confirm that plasmid DNA was extracted
role of gel electrophoresis
restriction enzymes – Hindii, bamhii
what was used to digest plasmid DNA
1. a semi-synthetic ampicillin analog
2. b-lactamase (bla) gene also confer resistance to carbenicillin
– more stable than ampicillin ie\ less likely to breakdown in presence of beta-lactaases
– reduce the growth of satellite colonines on plates during long-term incubations
1. what is carbernicillin
2 why use carbenicillin istead of ampicillin
An autonomous, self-replicating, extrachromosomal DNA mole
T. plasmid
Incorporating a DNA molecule into a chromosomal site or a cloning vector
T. cloning
A population of cells that carry a cloning vehicle with the same DNA insert
T. Clone
The *uptake of extrachromosomal DNA* in a bacterium or yeast cell in
which the DNA often *changes the phenotype* of the recipient organism
T. Transformation
A cell which has *taken up extrachromosomal DNA* and is expressing a
change in *phenotype*
T. Transformant
Cells which have been treated by chemical or physical means in order
to be sensitive to foreign DNA
T. Competent cells
bluewhite selection technique to select colonies with a successful insert (kanamycin resistance gene)
indirect selection method
Plate 100 μL from tube 3 onto 3 LB/carb/kan plates
direct selection method

Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall.

EDTA or Tris
– chelates (carries) divalent cations in the solution preventing DNases from damaging the plasmid
– destabilizing the cell wall

– maintains the osmotic pressure so the cells don’t burst, also pH

– RNase A degrade cellular RNA when the cells are lysed. –> we want to keep the DNA plasmid

TP. re-suspended in a solution of sucroseTris or EDTA – solution 1



– detergent
– break down cell membrane
-denatures cell proteins –> help w later separation of proteins from plasmid

[break down cell wall] alkaline lysisdenaturation – disrupts H-bonds between DNA –> double-stranded plasmid DNA to single stranded DNA (ssDNA)

TP. SDSNaOH – solution II

Solution becomes more basic –> *hydrogen bonding re-established* –> *ssDNA re-nature to dsDNA* ie\ circular plasmid DNA re-nature

bacerial genomic DNA (gDNA) is very long relative to plasmid DNA –> plasmid DNA reforms much easier and faster than gDNA –> selective part.

impossible to properly anneal those huge gDNA stretches.

–> EXPERIMENTAL ERROR: This is why it’s important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep.

TP. KOAc – potassium acetate

regarding g-DNA

separated from the plasmid DNA solution by centrifugation.

*double-stranded plasmid* *dissolve* easily *in solution*,

single stranded DNA + SDS + denatured cellular proteins stick together through hydrophobic interactions and precipitate

TP. White precipate

Add 150 μL of ice-cold Solution III (KOAc- potassium acetate) to each tube. Close caps and mix by rapid inversion several times.

Let stand on ice for 10 minutes. A white precipitate should form.

plasmid DNA separated from the majority of the cell debris

solution highly contaminated – salt, EDTA, RNase, residual cellular proteins and debris –> not much use, need to purify more

TP. supernatant (what is present in the supernant)

Transfer 400 μL of supernatant to a clean labeled tube. Avoid pipetting precipitate

organic solvent

concentrate and precipitate the plasmid DNA from the supernatant to be isolated

clean up the solution

TP. isopropanol
nucleic acid operations
TP. TE buffer
Purification of DNARNA
TP. Zymo-Spin columns and collection tubes
E. coli
What bacterial cultures were used
Remove 5′ phosphates from the ends of DNA vector that have been cut with a RE

5’P-DNA –> 5’OH-DNA + Pi

prevents self-ligation (recirculization) of the vector in subsequent ligation reactions –> facilitates ligation of other DNA fragments into the vector (e.g. subcloning).

Alkaline phophatase (AP) catalyzes what reaction
transform a strain of E. coli DH5a to be resistant to both ampicillin and kanamycin by creating recombinant pUC18 plasmid by restriction digestion and cloning
Purpose of the whole experiment
confirm that plasmid DNA was extracted
day 2:

why use agarose gel electrophoresis

pKAN – 4194 bp
pUC 18 – 2686 bp
pKAN will appear above
pKan plasmid and pUC18 plasmid which one will be on top on the gel
1. different plasmid topologies =
plasmid conformations

6. Nicked
supercoiled (thicc)
circular, single-stranded

3. structure deviates from the theoretical “circular” plasmid molecule

4a. treated lysate too vigourously
4b. digestion of the sample with EcoRI
4c. smear in contrast to linear band after digestion of multimeric plasmid forms

5. supercoiled

[1. what does [multiple visible bands ovarying intensities and migration observed folloing the alkaline lysis procedure] mean}

6. summarized of 4 types order (top to bottom on gel)

3. what do these share in common

4. sample contaminated with bacterial chromosomal DNA
4a. how does it formed
4b. how to identify
4c. how does it look like

5. what type are we interested in]

1. fused products of several plasmids recombined together
1a.several times larger than the individual plasmid –> run very slowly in agarose regardless of their conformation
1. what are multimers
1a. How are they on the agarose
1a. interested in this one
1b. (pUC) lower band compares to circular (also pUC) (migrates faster than predicted)
1c. (pTZ19) multimeric forms of supercoiled plasmid DNA
– a tiny bit above the pUC supercoiled
– not genomic DNA
– linearization using restriction digestion, genomic DNA –> single defined band with the size of the linearized (plasmid monomer) form

3a. denatured supercoiled DNA
– very weak band below pUC supercoiled

2a. native conformation found in vivo
2b. extra twists in the double helix strand.*

supercoiled plasmid

1a. with respect to study
1b. pUC position on the gel and migration rate
1c. mutimeric form
– position on the gel
– regarding DNA
– what to do to distinguish, from what –> result

3a. the bad form
– position on the gel

2a. what is it*
2b. formation*

1c. relaxed circular plasmid
1a. during replication, cell topoisomerases nick on strand of the DNA helix and relax the superhelical tension, allowing polymerases to gain access to the DNA
1b. slowest migrating form in an agarose gel
1. nicked
1c. what is it *
1a. when and how does it formed *
1b. migration rate
1a. (pTZ19) linearized form
– same with (pUC) open circular form
1b. after restriction digestion with EcoRI –> DNA helixplasmid is cut in both strands at the same place
1c. nuclease contamination or harsh treatment during purification
1. linear plasmid (forms)
1a. form on gel
– position on gel
1b. forms how
1c. lab error that leads to this
1a. (pUC) open circular form
– upper band compares to supercoiled (pUC)
– ds DNA
– nicked in one of the strands to released supercoils

1b. harsh alkaline lysis step bc incubated for too long

1c. DNA become permanently denatured resulting in single stranded closed circles

1. Circular, single stranded plasmid

1a. form see on gel
– position on the gel
– characteristics
– how to turn into supercoil

1b. how does it form*
1c. result


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