21/10/16 Mohammad Yousaf

BIO255H1F 1002536641

Assignment 1: Lab Report

Introduction:

The ability of plants to deal with drought conditions has become increasingly

important due to global climate change. In this study, the plant model

species Arabidopsis thaliana was used to study a specific gene that is

upregulated shortly after the onset of drought like conditions. The

Arabidopsis plant is excellent for such studies as its genome has been fully

sequenced and it has a short life cycle (1).

The specific gene studied was At5g24420 PGL5 (420). It Is a gene that has

been shown to code for an enzyme similar to glucosamine (1). Thus the

protein may function in a pathway that involves the metabolism of

carbohydrate. In this study, the specific gene sequence will be confirmed and

then the protein and its metabolic pathway will be studied to elucidate the

immediate drought response in Arabidopsis.

The first step in the study was to isolate the gene of interest from the plant

cell. Thus, a DNA extraction protocol was followed to isolate all of the plants

DNA from the other cellular components. Thereafter, the specific gene of

interest was amplified using the polymerase chain reaction (PCR). The PCR

products were then digested and inserted into a phagemid vector DNA,

which was inserted into bacteria cell. The gene was replicated in bacteria

and thereafter isolated via a DNA-mini Prep protocol. A negative result was

obtained and the gene of interest was not fully isolated from the bacteria.
21/10/16 Mohammad Yousaf
BIO255H1F 1002536641

Methods and Materials:

Young Arabidopsis leaf tissue was obtained and ground to a fine powder. A

series of buffers and reagents were added followed by centrifugation to

separate the cellular components (cell wall membranes, proteins) from the

DNA. The DNA was then further purified by washing with isopropanol and

ethanol and then drying. Finally, the DNA was stored in TE buffer.

The specific region of DNA containing the 420 gene was then selectively

amplified via PCR. A master mix solution was prepared with the following

components: PCR buffer, MgCl2, dNTPs, Forward Prime, Reverse Primer, Taq

DNA polymerase, distilled water. The specific primers ensured that the

correct region of DNA was amplified by the polymerase. A positive control

and negative control (distilled water) were used to ensure results were

accurate.

The amplified DNA (PCR product) was placed in a restriction buffer and

digested using BamHI and EcoRI at 37*C. Similarly, phagemid DNA was also

digested with BamHI and EcoRI. This allowed complementary ends of the

phagemid DNA to bind the PCR DNA. The digest products were mixed and

DNA ligase was added to covalently join the gene of interest to the

phagemid. A culture of E. coli bacteria was obtained and the ligation solution

(phagemid with 420 gene) was added. The cells were heat-shocked for 20

seconds and then stored in LB medium on an agar plate.

21/10/16 Mohammad Yousaf
BIO255H1F 1002536641

Bacterial colonies with the gene of interest were obtained and mixed with

various DNA Mini-Prep buffers and then centrifuged. The DNA solution was

placed in a Silica column and the DNA vectors were isolated by elution.

Results and Discussion:

21/10/16 Mohammad Yousaf
BIO255H1F 1002536641

Fig

ure 1: Results from the Restriction Enzyme Digest of various controls and the

420 gene phagemid.

21/10/16 Mohammad Yousaf
BIO255H1F 1002536641

The diagnostic gel electrophoresis includes six lanes, each with a mixture

solution that has a specific function. The DNA ladder solution provided a

reference as to the relationship the size of genomic DNA and the distance

travelled. The 420 gene digest lane is the lane with digested vector with the

gene of interest. The positive control shows what the expected results should

have been if everything proceeded correctly. The negative controls may help

with determining where an error may have occurred if a negative result was

obtained, as was the case.

No bands were observed in either the 420 Gene Digest or the Undigested-

Vector, this suggests that an error may have occurred in the previous step

where the Phagemid vector was extracted from bacteria. It is possible that

the phagemid DNA did not bind to the silica membrane in the column and

hence, no DNA eluted in the end. To confirm this was the issue, replicate

protocols should have been set up for the Mini-prep protocol.

References:

1. Cordon A, Neumann M. Arabidopsis DNA Isolation. In: Neumann M, Maharaj N,

editors. BIO255H Molecular Biology Course Manual. Toronto: University of

Toronto Press, 2016. p 1-2 1-13, p 2-1 2-11.