INTRODUCTION

The terms "recombinant DNA technology," "DNA cloning," "molecular cloning," and
"gene cloning" all refer to the same process: the transfer of a DNA fragment of interest from
one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of
interest can then be propagated in a foreign host cell. This technology has been around
since the 1970s, and it has become a common practice in molecular biology labs today.
Scientists studying a particular gene often use bacterial plasmids to generate multiple
copies of the same gene. Plasmids are self-replicating extra-chromosomal circular DNA
molecules, distinct from the normal bacterial genome (Figure 1). Plasmids and other types of
cloning vectors were used by Human Genome Project researchers
to copy genes and other pieces of chromosomes to generate
enough identical material for further study.

Figure 1
To "clone a gene," a DNA fragment containing the gene of interest is isolated from
chromosomal DNA using restriction enzymes and then united with a plasmid that has been
cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined
with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following
introduction into suitable host cells, the recombinant DNA can then be reproduced along with
the host cell DNA.

Plasmids can carry up to 20,000 bp of foreign
DNA. Besides bacterial plasmids, some other
cloning vectors include viruses, bacteria artificial
chromosomes (BACs), and yeast artificial
chromosomes (YACs). Cosmids are artificially
constructed cloning vectors that carry up to 45 kb of
foreign DNA and can be packaged in lambda phage
particles for infection into E. coli cells. BACs utilize
the naturally occurring F-factor plasmid found in E.
coli to carry 100- to 300-kb DNA inserts. A YAC is a
functional chromosome derived from yeast that can
carry up to 1 MB of foreign DNA. Bacteria are most
often used as the host cells for recombinant DNA
molecules, but yeast and mammalian cells also are
used.




THODOLOGY
DNA Extraction, Purification & Quantification
Spin 1.5ml culture for 5 min full speed. Decant supernatant.

Add 300ml lysis solution, vortex 2-5 second.

Add 2ml proteinase k and incubate at 37°C for 30 minutes.

Add 500ml PCÌA. Mix well

Spin 13000rpm for 3 min. Transfer first layer into new tube.

Add 1/10 volume Sodium Acetate.

Add 100% Etoh until full,gently mix it.

Ìncubate at -70°C for 15minutes.

Spin for 5 min at 13000rpm.Discard supernatant.

Add 500ml of 70% cold Etoh. Resuspend the pellet.

Spin 10000 for 2 min.

Dry pellet in LAF.

Dissolve in 30ml TE buffer

Ìncubate 37°C for 15min and store at 20°C
DNA Purification
5ml of DNA solution+495ml dilute distilled water. Vortex.

Read absorbance at 280 nm. Read the reading. Calculate concentration of DNA in the
solution. Check the DNA purification.

Primer Design by using on-line Bioinformatics Tools
DNA Amplification
Pipette and mix all the following reagent in a tube on ice.
PCR Reagent Final Concentration Volume(ml)
5x PCr buffer 1x 5 15
2mM dNTP's mix 200µm 2.5
50mM MgCl
2
3mM 1.5
15µM Primer forward 0.3µM 0.5
15µM Primer reverse 0.3µM 0.5
Template DNA 200ng 3
5U/ml taq polymerase 0.5U 0.1
DdH
2
O Up to 25µl 14.4


Short spin, place the PCR tube in the PCR machine.

Competence Cells Preparation& Stock cultures Preparation
Stock culture
Microcentrifuge 850ml culture E.coli+150 ml glycerol.
Competence cell
E.coli structure centrifuge 5 min at 5000rpm at 4°C. Discard supernatant.

Add 5 ml CaCl
2
. Dissolved pellet by pipetting.

Ìncubate on ice for 20 minutes.

Centrifuge 5000rpm at 4°C for 5 min. Dicard supernatant.

Add 1 ml CaCl
2
. Dissolve pellet.Transfer new microcentrifuge tube.

Spin down 5000rpm for 5 min at 4°C.Pipette out 800ml.

Dissolve pellet in remaining 200ml.

Ligation of PCR Product into pGEM-T Vector & Transformation of Escherichia coli.

Thaw competent cell on ice.

Flicking to mix.

Add 10ml ligation mixture.

Flicking to mix.

Ìncubate on ice 45mins.

Heat shocked 42°C,2mins.

Return to ice 3 min.

Transfer to SOC tube.

Ìncubate 37°C,1hour,150rpm.

Centrifuge at 4000rpm,5min.

800ml cell resuspended

Plate on 2 LB/ampicilin plate(120ml,80ml)

Allow it to dry.

Ìncubate overnight at 37°C.
Plasmid Extraction(Miniprep)
Harvest bacteria,spin 9000rpm for 5 min.

Remove supernatant,resuspend pellets with 50ml solution 1.

Add 100ml solution 2,mix,incubate on ice for 3 min.

Add 75ml solution 3,vortex,incubate on ice,5 min.

Transfer mixed lysate to eppendorf tube, spin 12000g,5min.

Transfer supernatant to new eppendorf tube

Add 1.5RNase, incubate at 37°C for 15 min.

Add equal volume=200ml phenol:chloroform, vortex, spin 12000g for 2 min.

Transfer supernatant to new eppendorf tubes.

Add 400ml absolute ethanol,vortex,spin 12000g for 5 min.


Discard supernatant,rinse pellet with 300ml ice cold ethanol,spin 12000g for 5 min.

Discard supernatant,air dried pellets.

Dissolve pellet with 50ml distilled water.
Restriction digestion.
Caculate amount of ingredients to be used.
Concentration
of DNA
Total
volume of
DNA to
make up
1µg
Volume of
EcoR1(1
unit)
BSA 10x Buffer Distilled
water
Total
reaction
volume
5µl 1µl 2µl 2µl 10µl 20µl


Mix all the ingredient.

Microcentrifuge at top speed,3seconds

Ìncubate 37°C for 1 hour

centrifuge






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"U$TION
DNA Extraction, Purification & Quantification
1.What is DNA,where it is located and what is the function?
DNA is deoxynucleotide acid that located in the cell nucleus. Ìt stored genetic information.

2.What are three steps in DNA extraction? Elaborate the three steps.
The three steps are homogenization,deproteinization and precipitation.................

3.Why does DNA rise to top after additiona of acohol?
DNA is insoluble in alcohol

4.How can you prove that the substance that you extract is DNA?


5.Assuming that you have extracted DNA, do you think you get pure DNA?Ìf yes, why?Ìf no
what are the contaminants that present and how to get rid of it?
Not pure.The contaminants that present are protein and RNA.The way to get rid of it is by
DNA purification.

DNA Amplification
1.What is PCR,function and its advantages in molecular research?
PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences.The
function is to allowed the generation of large amount of a single DNA sequence from a
mixture of sequences.

2.What are the potential problems that can inhibit amplication?
.......



Competence Cells Preparation & Stock cultures Preparation.
Stock Culture Preparation
1.Explain what the aseptic technique is and how to avoid cross contamination occur during
stock preparation?
Aseptic technique is a method of sterilizing by handling bacteria culture in laminar flow and
wipe up the laminar flow with 70% ethanol.

2.Define the function of glycerol in culture stock?
....

3.How many types of stock culture can be done for bacterial cell?
...
Competence Cells Preparation
1.What are three basic steps in the introduction of plasmid DNA into cells?
Preparation of competence cells, transformation of competence cell and selection of
transformants.

2.What are the meaning of 'competence'?
The ability of some bacterial strains to take up 'naked' DNA from the surroundings.

3.Explain shortly the purpose of competence cell in transformation stage.
....

4.Explain the function of CaCl
2
in the preparation of competence cell.
To facilitate the entry of plasmid DNA vectors into E.coli in conjunction with structural
alteration of the bacterial cell wall.




Ligation of PCR Product into pGEM-T Vector & Transformation of Escherichia coli.
1.List 5 factors that effect transformation efficiency?
Plasmid size, quantity, purity, DNA configuration (linear, closed circular,nicked) and
selectabe marker.

2.What are the reasons for the low level of transformation during the experiment?
High level of DNA

3.What are other methods of transformation used for plant and yeast?
.....

4.Why in this experiment we used LB/ampicilin plate to plate out the transformants?
To screen the existent of E.coli as it contain ampicilin resistant.

Plasmid Extraction (Miniprep)
1.Explain what is the meaning of miniprep plasmid extraction?
Preparing partially purified plasmid DNA in small quantities from number of transformants.

2.What are other methods to extract plasmid besides miniprep pasmid extraction?
.....

3.Explain 3 basic steps involve in plasmid DNA isolation?
Growth of bacteria and amplification of the plasmid, harvesting and lysis of the bacteria and
purification of the plasmid DNA.

4.Give two major differences between E.coli DNA and plasmid DNA.
i)the E.coli chromosome is much larger than the DNA of plasmids used as vectors.
ii)The bulk of E.coli DNA extracted from cells is obtained as broken, linear molecules,By
contrast, most plasmid DNA is extracted in a covalently closed,circular form.

Restriction Digestion
1.Prepare a reaction mixture for a restriction digest containing components from the
following stock solutions:DNA(2 mg/ml),10x reaction buffer and ,2(10 U/ul).Ìndicate the
minimum total reaction volume required for digesting 10µg of plasmid DNA and the order in
which these reagents should be added .
......

2.Ìn your opinion how to make sure your DNA fragment is fully and completely digested?
.....

3.Why bovine serum albumin(BSA)is sometimes needed during restriction digestion?


4.How to handle ethidium bromide solution safelky and what are the consequences to the
exposure of this chemical compound.

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