7/30/2019 TEAM 3333333333333

1/13

JEFFREY

MARISKA

SHALINI

SARAH

=D

7/30/2019 TEAM 3333333333333

2/13

What are pure cultures?

Pure culture is a lab culture containing a

single species of organism.

7/30/2019 TEAM 3333333333333

3/13

What is a colony?

A cluster of bacteria.

7/30/2019 TEAM 3333333333333

4/13

Our results

Record your results in the table below.

Plate Ligation components Growth Transformation No. of colonies

1 Teams PCR product

and plasmid

Yes/No Yes/No 2

Negative control no no 0

Competency of cell = number of colonies/ amount of DNA (ug)

= 605/0.001

=6.05 x 10^5 cells per ug of DNA

Our transformation efficiency

= 2/ 0.001

= 2000

= 2000 cells/ g

7/30/2019 TEAM 3333333333333

5/13

always indicative of a

successful transformation?

Explain your answer No, because our plates could be contaminated

by other microorganisms which are also

resistant to ampicillin. Also Plates with weak or inactive antibiotic will

also allow growth of bacterial colonies eventhough the colonies do not contain a drug-

resistance plasmid. Satellite colonies, which are not resistant to the

ampicillin are also able to grow on the plate asthey grow around the ampicillin resistant colony

7/30/2019 TEAM 3333333333333

6/13

which bacteria colony (a) or (b)

would you select to inoculate

your culture?

Bacteria colony (b) because bacteria colony (a) are just satellite colonies (non-

antibiotic resistant) which are just tiny colonies growing around the antibiotic resistant

colony (The satellites form because the beta-lactamase released by the bla-expressing colony degrades the ampicillin in the vicinity of the colony.)

7/30/2019 TEAM 3333333333333

7/13

preparing volumes for 4

reactions of PCR master mix

solutions? To ensure that there is enough master mix to

make up for any pipetting errors or other

mistakes during the experiment and to savetime and minimize error in the repeating

pipetting steps for small volumes of the same

solutions.

7/30/2019 TEAM 3333333333333

8/13

conducting Colony PCR and

are there advantages to this

technique? Colony PCR is can be used after a

transformation to screen colonies for the

desired plasmid. The primers used in this reaction is used to

generate a PCR product of known size, Thus,colonies which give rise to an amplification

product of the expected size are likely to containthe correct DNA sequence.

The advantage is time is saved by avoiding thegrowth of overnight cultures and subsequent

plasmid DNA isolation

7/30/2019 TEAM 3333333333333

9/13

Why was ampicillin added to

the growth medium? Its to isolate the colonies which are resistant

to ampicillin and to let us know if the bacteriahas taken up the recombinant plasmid whichcontains the gene of interest.

Thus, if bacteria are able to grow in the growthmedium, it indicates that they have taken upthe plasmid which contains the ampicillinresistant gene

Ampicillin is a selective media. It is added so

that only certain bacteria can grow. This is toprevent other cells that do not have suchresistance to grow in the media.

7/30/2019 TEAM 3333333333333

10/13

What further analysis may be

performed on the selected

bacterial clones in order to confirmthe presence of the cloned insert?

We can use gel electrophoresis to see if the

bands match our previous PCR product. If

the gene is inserted, then the bands should

be a match and should have the same

sequence and size.

7/30/2019 TEAM 3333333333333

11/13

a) Upon adding NaOH/SDS (step

4), why is mixing done by hand

(inversion) instead of votexing? In step 3: GTE is used to resuspend bacterial cell pellets prior to

lysing (breaking open) the cells and harvesting the plasmid DNAinside.

Achieving a homogenous suspension of whole cells during thisstep so that the subsequently added lysis solution can get to allof the cells is key to getting good DNA yields. GTE is designedto do this while also providing a stable environment for the DNA.

By vortexing, it can shear the bacterial chromosome, leavingfree chromosomal fragments in the supernatant which will co-purify with the plasmid DNA.

7/30/2019 TEAM 3333333333333

12/13

the incubation MUST NOT

exceed 5 mins before going to

step 5 (adding potassiumacetate). Why is this so? If left for too long, the Plasmid DNA strand

will denatured to the point where the plasmidDNA will become single stranded and the

restriction enzyme will not be able to cut it.

7/30/2019 TEAM 3333333333333

13/13

c) How are proteins removed and

why is it important that your

plasmid must be free of proteincontamination? Protein is isolated from the plasmid DNA

using PCIA (phenol/chloroform/isoamylalcohol) by adding about 0.3 ml.

It must be free from protein contamination as

the protein could possibly degrade the DNAand inhibit PCR.