JEFFREY MARISKA SHALINI SARAH =D

What are pure cultures?

Pure culture is a lab culture containing a single species of organism.

What is a colony?

A cluster of bacteria.

Our results
Record your results in the table below. Plate 1 Ligation components Teams PCR product and plasmid Negative control Growth Yes/No no Transformation Yes/No no No. of colonies 2 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

Are colonies on an agar plate 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 even though the colonies do not contain a drugresistance plasmid. Satellite colonies, which are not resistant to the ampicillin are also able to grow on the plate as they grow around the ampicillin resistant colony

which bacteria colony (a) or (b) would you select to inoculate your culture?

Bacteria colony (b) because bacteria colony (a) are just satellite colonies (nonantibiotic resistant) which are just tiny colonies growing around the antibiotic resistant colony (The satellites form because the beta-lactamase released by the blaexpressing colony degrades the ampicillin in the vicinity of the colony.)

What is the reason for 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 save time and minimize error in the repeating pipetting steps for small volumes of the same solutions.

What is the purpose of 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 contain the correct DNA sequence. The advantage is time is saved by avoiding the growth of overnight cultures and subsequent plasmid DNA isolation

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 bacteria has taken up the recombinant plasmid which contains the gene of interest. Thus, if bacteria are able to grow in the growth medium, it indicates that they have taken up the plasmid which contains the ampicillin resistant gene Ampicillin is a selective media. It is added so that only certain bacteria can grow. This is to prevent other cells that do not have such resistance to grow in the media.

What further analysis may be performed on the selected bacterial clones in order to confirm the 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.

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

By vortexing, it can shear the bacterial chromosome, leaving free chromosomal fragments in the supernatant which will copurify with the plasmid DNA.

the incubation MUST NOT exceed 5 mins before going to step 5 (adding potassium acetate). Why is this so?

If left for too long, the Plasmid DNA strand will denatured to the point where the plasmid DNA will become single stranded and the restriction enzyme will not be able to cut it.

c) How are proteins removed and why is it important that your plasmid must be free of protein contamination?
Protein is isolated from the plasmid DNA using PCIA (phenol/chloroform/isoamyl alcohol) by adding about 0.3 ml. It must be free from protein contamination as the protein could possibly degrade the DNA and inhibit PCR.