Department- School of Biotechnology

Stream- MSc in Microbiology

Subject- Lab on Genetic Engineering (MSUMC-291)

Submitted on- 10.06.20221.

Ankan Kr. Maity

Roll No-30028121027

Reg. No-213001828110027

1st Year , 2nd Sem

SL Name of the Experiment Page No Date of Signature
NO practical

1 LB media preparation

2 Isolation of Bacterial strain

3 Generation of Competent cells using

the CaCl2 method

4 Bacterial transformation using heat

shock method

5 Alkaline lysis Miniprep of plasmid DNA

6 Agarose gel electrophoresis of plasmid

7 Restriction enzyme activity

8 PCR method
Exp-1 LB Media preparation protocol

Introduction

This protocol makes 500 mL of broth or ~25 plates. In order for bacteria to be successfully cultured,
they must be grown in the appropriate media. LB, also known as Luria Bertani, is a nutrient rich
broth that is a standard for culturing Escherichia coli, as it allows for quick growth and high yields.
Therefore, the proper preparation of LB will be crucial to maintaining our bacterial stock throughout
the summer. Furthermore, addition of agar to LB broth creates a gel for bacteria to grow upon, and
is therefore used for plating bacterial cultures on petri dishes.

Materials and Reagents

⚫5 g Bacto-tryptone

⚫2.5 g yeast extract

⚫5 g NaCl

⚫7.5 g agar (Only necessary if making LB agar plates)

⚫500 mL of dH2O (distilled water)

Procedure:

1. Mix, autoclave at 15-20 psi at 150-120°C for 20 mins (use conical flask of a double

volume).

2. Wait till temperature of media comes down to -60°C, pour agar solution maintaining sterile
conditions.

3. Label agar plates at the bottom accordingly with dates.

4. Store plates in 4°C (refrigerate) or test for contamination (single plate O/N at 37°C)

Observation:
Exp-2 Isolation of DH5a and XL1Blue bacterial strains

Introduction

In many applications of microbiology it is necessary to purify one type of bacterium from a diverse
population of many different types. Often the starting culture will be a mixed population of different
species or strains of the same species. In genetics, it is often desirable to have a pure culture of
bacteria to ensure that the genetic make-up of all cells in a culture is uniform. The easiest way to do
this is to establish a clonal population of cells. In other words, grow the culture from a single cell.
Because all cells of the culture will have descended from the same cell, the vast majority of cells in
the culture will be genetically identical (only those that have acquired a spontaneous mutation, a
rare event, will be different).

To isolate a single cell, bacteria are essentially diluted on a petri plate by successive streaking The
resulting isolated single cells then grow to forma visible colony that is "picked" and used to start a
culture.

Protocol

1. With your marker pen, label the plate with your initials, the date, and the strain name.

2. Draw two lines on the bottom of the agar plate to divide the plate into four quadrants as in the
picture below.

3. Flame the inoculating loop with the Bunsen burner, then cool the loop by pressing it into the agar
10 or so times, pausing for one second in the agar each time. Pick up a small amount of cells (e.g.
DH5a and XL1Blue) from respective plates and start streaking the culture in one quadrant of the agar
plate (properly labeled) as in the picture shown below. Remember to flame the loop before
streaking from quadrant to quadrant

4. At the end of the procedure, you should have all four quadrants streaked on the plate.

5. Incubate the plates inverted at 37°C sealed with parafilm.

6. The next day, you should expect to see a few pure colonies appear in the third and fourth

quadrants as shown in the picture below.

Exp-3 Generation of Competent cells using the Calcium
Chloride Method & Bacterial Transformation

Introduction

Bacteria which are able to uptake DNA are called "competent" and can be made so by treatment
with calcium chloride in the early log phase of growth. Cells of a liquid culture are grown to an
exponential phase (mid log phase, OD com 0.45-0.55) and pelleted, followed by the removal of the
supernatant and resuspension in ice-cold 100mM CaCl2 to make a dense suspension. A bacterial cell
that is growing exponentially contains several holes in the cell wall. These holes are sites of
extension, or growth of the murein sacculus cell wall. When cells that are growing exponentially are
put onto ice, cellular activity (including the repair and consequent growth of the cell wall) is greatly
slowed and these holes remain open. theory is that these holes are necessary for entry of the DNA
into the cell.

Experimental considerations

1. This method is easy and fast, and provides reasonable transformation efficiencies (10^5-10^6
colonies per ug of DNA) which are sufficient for routine subcloning experiments.
2. Frozen vials can be kept at -80°C for months-years without significant loss of viability or
efficiency

Protocol

Preparation of Competent cells

1. Inoculate one colony into 5 ml LB medium.

2. Incubate overnight on shaker at 37°C.

3. Inoculate 1ml of the overnight culture into 100 ml of pre-warmed LB in a 500 ml conical flask
(1:100 dilution)

4. Incubate on shaker at 37°C for 2 to 4 hours (Check OD at 1 hour gap for checking absorbance

with LB medium as blank). Grow cells till OD reaches the mid log phase (O.D at 600nm 0.45-0.55)

5. Spin cells for 10 minutes at 3000 to 4000 rpm at 4°C.

6. Resuspend cell pellet very gently in 15 ml ice cold 100 mM CaCl2 (Solution to be sterile before
use).

7. Incubate for 30 minutes on ice.

8. Spin cells for 10 minutes at 5 K rpm at 4°C. Repeat the steps 6 and 7 for two more times

(preferably).

9. Resuspend cell pellet very gently in 2 ml ice cold 100 mM CaCl2.

10. Add sterile glycerol to a final concentration of 15 to 20% (or prepare 100 mM CaCl2 + 15 to 20 %
sterile glycerol) [Glycerol as a cryoprotectant depresses the freezing point of bacterial cells,
enhancing supercooling. It does so by forming strong hydrogen bonds with water molecules,
competing with water-water hydrogen bonding. This disrupts the crystal lattice formation of ice
unless the temperature is significantly lowered).

11. Aliquot 0.1 ml per eppy tube (microtube) on ice [keep tubes on ice until transferring to -80°C)

Quick chilling in liquid N2 or Dry Ice in Ethanol Bath is preferred for increasing competency.

12. Immediately transfer aliquots to -80°C freezer.

Observation:
Exp-4 Bacterial Transformation using heat shock method
and Antibiotic Selection

Introduction

Bacterial transformation is the process by which bacterial cells take up naked DNA molecules. If the
foreign DNA has an origin of replication recognized by the host cell, the bacteria will replicate the
foreign DNA along with their own DNA. When transformation is coupled with antibiotic selection
techniques, bacteria can be induced to uptake certain DNA molecules, and those bacteria can be
selected for that incorporation. Bacteria which are able to uptake DNA are called "competent and
can be made so by treatment with calcium chloride in the early log phase of growth. Cells of a liquid
culture are grown to an exponential phase (mid log phase, OD 600nm-0.45-0.55) and pelleted,
followed by the removal of the supernatant and resuspension in ice-cold 100mM CaCl2 to make a
dense suspension A bacterial cell that is growing exponentially contains several holes in the cell wall.
These holes are sites of extension, or growth of the murein sacculus cell wall. When cells that are
growing exponentially are put onto ice, cellular activity (including the repair and consequent growth
of the cell wall) is greatly slowed and these holes remain open. One theory is that these holes are
necessary for entry of the DNA into the cell.

The bacterial cell membrane is permeable to chloride ions, but is non-permeable to calcium ions. As
the chloride ions enter the cell, water molecules accompany the charged particle. This influx of
water causes the cells to swell and is necessary for the uptake of DNA. The exact mechanism of this
uptake is unknown. It is known, however, that treatment with heat increases the efficiency of
transformation. When E. coli cells are subjected to 42°C heat, a set of genes are expressed which aid
the bacteria in surviving at such temperatures This set of genes is called the heat shock genes. The
heat shock step facilitates the uptake of DNA, but the exact mechanism is unknown. At
temperatures above 42°C, the bacteria's ability to uptake DNA becomes reduced, and at extreme
temperatures the bacteria will die.

After the heat shock step, intact plasmid DNA molecules replicate in bacterial host cells. To help the
bacterial cells recover from the heat shock, the cells are briefly incubated with non-selective growth
media. As the cells recover, plasmid genes are expressed, including those that enable the production
of daughter plasmids which will segregate with dividing bacterial cells. However, due to the low
number of bacterial cells which contain the plasmid and the potential for the plasmid not to
propagate itself in all daughter cells, it is necessary select for bacterial cells which contain the
plasmid. This is commonly performed with antibiotic selection. E coll strains such as DH5a are
sensitive to common antibiotics such as ampicillin, kanamycin, and chloramphenicol. Plasmids used
for the cloning and manipulation of DNA have been engineered to harbor the genes for antibiotic
resistance. Thus, if the bacterial transformation is plated onto media containing antibiotics, only
bacteria which possess the plasmid DNA will have the ability to metabolize the antibiotic and form
colonies. In this way, bacterial cells containing plasmid DNA are selected.
Experimental considerations

This method is easy and fast, and provides reasonable transformation efficiencies (10^5-10^6colonies
per µg of DNA) which are sufficient for routine subcloning experiments.

Protocol

1. Thaw a vial of competent cells on ice (DH5a or XL1 Blue) for 5 mins.
2. Dispense 100 µl of competent cells in fresh microtube.
3. Add 1 µl of plasmid DNA, [a) pUC19 vector, conc. 0.05 µg DNA or 1.0 µg DNA or any plasmid
vector with or without insert) (mix by tapping with finger).
→To make the plasmid DNA available to the bacteria
4. Incubate for 30 minutes on ice.
→To allow the uptake of DNA
5. Heat shock: Place tubes at 42°C water bath for 90 seconds.
→Heat shock may promote water movement that is necessary for uptake of the DNA
6. Immediately transfer tubes to ice for 5 minutes.
→Again, the change in temperature may promote movement of water that promotes the movement
of DNA into cells.
7. Add 0.9 ml LB (with no antibiotic) and mix briefly
8. Incubate samples in 37°C in a rotary shaker for 45 min to 1 hr.
→To allow cell repair and expression of genes of the plasmid whose presence will be selected.
9. Plate 0.05-0.1 mi culture onto an ampicillin containing LB plate.
→To select for growth of the cells that have received the plasmid.
10. Incubate plate overnight at 37°C, inverted and sealed with parafilm
11. Check the growth of the selected colonies and store in a refrigerator at 4°C or proceed for
estimation of Transformation Efficiency

Observation:

Figure 1 without ampicillin(control)

Figure 2 with ampicillin
Determination of Genetic Transformation Efficiency
Transformation Efficiency Definition

The transformation efficiency of bacteria is calculated as successful transformants (cfu) per

microgram (µg) plasmid DNA

Calculation
(Wavelength in nm) OD
200 0.340
210 0.240
220 0.131
230 0.080
240 0.092
250 0.123
260 0.132
270 0.107
280 0.073
290 0.039
300 0.016
310 0.006
320 0.003
330 0.002
340 0.002
350 0.001
 Exp-5 Alkaline lysis Miniprep of Plasmid DNA
Aim:
Alkaline lysis miniprep of plasmid DNA.

Principle:

Plasmids have become indispensable tools for amplifying specific pieces of DNA and introducing these pieces
of DNA into bacterial cells. After a plasmid has been amplified by replication ina growing culture of bacteria, it
must first be purified away from the cells before it can be manipulated using the tools of molecular biology.
Plasmid mini-preps are used to quickly isolate small quantities of plasmids from bacterial cells. There are several
mini-prep protocols, but one of the most popular is the alkaline lysis method due to the high purity of the
resulting plasmid DNA. The method relies on the membrane-solubilizing properties of the zwitterion sodium
dodecyl sulfate (SDS) and high pH to break open cells and solubilize the cellular components. In subsequent
steps, each of the major componentsof the cell is removed from the sample until nothing but the plasmid
DNA remains. For instance, phenolis used to remove proteins, differences in solubility and centrifugation are
used to remove membrane constituents and associated chromosomal DNA, and RNA is degraded to its
constituent ribonucleotides, which do not precipitate with plasmid DNA in isopropanol.
Safety note: Phenol is a strong oxidizer that damages tissue (ie. skin, eyes, etc) on contact. Wear gloves and
work under a hood when handling phenol. Excessive amounts of water should be used to remove phenol from
exposed skin, and phenol soaked clothing must be removed immediately.
Materials required and preparation of stock solutions:
Bacterial Strain: (Store in -800C)
Transformed DH5α
Alkaline Solution 1 (50ml): (Store in 40C)
50mM Glucose
25mM Tris HCl (pH 8)
10mM EDTA (pH 8)
Alkaline Solution 2 : (Store at Room Temperature)
0.2N NaOH (Freshly diluted from 10N stock)
1% (w/v) SDS
Alkaline solution 3 (50ml) pH 5.2 (Store in 40C)
3M Sodium Acetate 60ml
Glacial Acetic acid 11.5ml
Water 28.5ml
Phenol-Chloroform-Isoamyl Alcohol Mix:
A mixture containing equal parts of equilibrated P:C:I (25:24:1) is frequently used to remove proteins from
preparation of nucleic acids. The chloroform denatures the proteins and facilitates the separation of
aqueous and organic phases and the Isoamyl Alcohol reduces the foaming during the extraction. For a
final volume of 50ml, 25ml of Phenol is added taking care not to disturb the Tris while extracting the Phenol
from its base. To this 24ml of Chloroform and 1ml ofIsoAmyl Alcohol is added. The solution is wrapped in
aluminium foil and kept at 4oC.

Protocol:

1) Inoculate one colony of DH5Alpha containing pGEMT-Easy/insert into LB tubes containing 3 ml LB media in
presence of appropriate antibiotics (ampicillin) and grow overnight in shaking at 37ºC.
→Antibiotics ensure that only the cells that have the plasmid will grow.

2.Transfer 1.5 ml of cells to a microfuge tube and pellet cells at 10,000 rpm for 2 min in the microfuge. After
centrifugation, the supernatant was decanted.
→This was done to collect cells and remove supernatant
Step 2 was repeated twice in the same microfuge tube to increase the yield.

Add 125 µl of Solution #1 (GTE) to the cell pellet and resuspend cells with a pipette.

→Glucose increases the viscosity of the solutions; Tris buffers the pH; and EDTA chelatesmetal ions, which are
required by most nucleases that degrade DNA.

200 µl of Solution #2 (NaOH/SDS) was added and the eppendorf inverted 7-8 times gently tomix and lyse cells.
→NaOH/SDS lyses the cells. SDS is a zwitterion that solubilizes membranes.

185 µl of Solution #3 (KOAc) was added and the tube inverted 7-8 times gently to precipitate cellmaterial.
→Acetic acid neutralizes the pH, and potassium precipitates the SDS and associated cellmembranes.

The vials were centrifuged for 10 minutes at 10,000 rpm at room temperature in a microfuge.

→This is done to pellet the cellular membranes and attached chromosomal DNA.

The supernatant is transferred to a new tube to avoid the pellet material.

→This is done to separate the plasmid DNA and protein from the membranes and chromosomalDNA.

An equal volume of phenol:chloroform:isoamyl alcohol (~400 µl) was added to 6 of the 10 tubes. The tubes were
vortexed for 10s.
→This is done to remove protein- many proteins are more soluble in phenol than in water.

To the other 4 tubes, 1 ml of ice-cold ethanol was added, followed by 1/10th volume (approximately 40 l) me of
3M NaOAc (pH 5.2) and the tubes vortexed well kept at -80ºC for half an hour.

The 6 tubes were centrifuged for 10 minutes at 10,000 rpm.

→This is done to separate the phenol and aqueous phases.

The upper aqueous phase is transferred to a fresh tube to avoid the protein interface.

Chloroform (~350 µl) was added to the aqueous phase of the 6 tubes and vortexed as above.

→Some proteins are more soluble in chloroform than phenol. Chloroform also removes residuephenol and provides
a cleaner interface.

Centrifuge 5 minute at 10,000 rpm for 10 minutes.

→This is done to separate the chloroform and aqueous phases.

Transfer the upper aqueous layer to a new tube.

2~3 vols ice-cold 100% ethanol was added to the aqueous phase followed by 1/10th volume of3M NaOAc
(pH 5.2), the tubes vortexed for 30 seconds, and incubate for 30 minutes at -80oC.
→This is done to precipitate the plasmid DNA.
The tubes were centrifuged for 10 minutes at 12,000 rpm.
→This is done to pellet the plasmid DNA.
The supernatant is removed and discarded, taking care to not disturb the small pellet which isthe plasmid DNA.
→This is done to remove the ethanol.

Add 500 µl 70% EtOH

→Washing with 70% EtOH removes salts that precipitate with the plasmid DNA in step 15.

Centrifuge at 12,000 rpm for 10 minutes.

→This is done to pellet the plasmid DNA.

Remove the EtOH taking care not to disturb the pellet.

The plasmid DNA is resuspended in 20 µl sterile water.

Observation:

Figure 2after addition of sol Figure 2 After addition of Sol2

Figure 3 after addition of sol 3 Figure 4 after addition of phenol

Figure 5 after addition of

Figure 6 after addition of sodium
chloroform
acetate
 Exp-6 Agarose Gel Electrophoresis of Plasmid
Aim:
To visualize DNA by Agarose gel electrophoresis.
Principle:
DNA can be separated by size and visualized by agarose gel electrophoresis. Agarose is a purified form of agar, which
is a polysaccharide purified from seaweed. Agarose gels are prepared by melting the agarose in electrophoresis buffer,
casting the desired size gel, and allowing it to polymerize as the mixture cools to form a uniform gel.

DNA is negatively charged due to its phosphate backbone and will migrate through the gel towards the anode (positive).
The electrophoresis buffer provides ions that carry a current and maintain the pH at a relatively constant value. The agarose
gel acts as a sieve that retards the migration of larger DNA molecules more than smaller ones. Hence, the DNA molecules
are separated according to size by the gel. To view the DNA, ethidium bromide (EtBr), which intercalates between the bases
of DNA, is used. Both DNA and EtBr absorb UV light. EtBr also fluoresces in the red/orange part of the light spectrum (590
nm) when excited by UV light, so the DNA/EtBr complexes can be viewed with UV light due to the increased concentration
of EtBr associated with the DNA.
Because EtBr is a carcinogen, contact should be avoided, gloves must be worn, and substances containing EtBr must be
disposed of properly. Above is a picture of a typical DNA agarose gel photographed under UV light with a UV filter to
remove the excess background UV light. Larger DNA molecules are retained at the top of the gel, while smaller pieces
migrate faster down to the bottom. Typically, DNA markers of known size are run on the same gel to allow an estimation of
the unknown bands by comparison.
THE ELECTROPHORETIC MIGRATION OF DNA THROUGH AGAROSE GEL IS DEPENDENTUPON 5 PARAMETERS:-
Molecular size or molecular weight : –
The rate of electrophoretic migration of DNA is inversely proportional to its molecularweight.
Rate log10 (molecular wt)
Thus shorter DNA fragments will move faster than the larger once.
Agarose concentration : –
The concentration of agarose is also an important factor on which the eletrophoretic migrationdepends. Mathematically
the relation is given by-
log µ = log µ0 – Krt
where
µ0=free eletrophoretic mobility
Kr = retardation co-efficient which is a constant depending upon of the gel, size andshape of migrating molecules
t= gel concentration
µ = electrophoretic mobility of DNA fragments in the gel

The table below gives an account of the various concentrations and the corresponding efficientrange of separation of
linear DNA molecules in kb :-

GEL CONCENTRATIONS EFFICIENT RANGE OF SEPARATION OF

(%) LINEAR DNA MOLECULES (kb)
0.3 60 – 5.0
0.6 20 – 1.0
0.7 10 – 0.8
0.9 7 – 0.5
1.2 6 – 0.4
1.5 4 – 0.2
2.0 3 – 0.1

The configuration of the plasmid DNA molecule :-

The configuration of the DNA molecule affects their mobilities. Three forms: covalently, closed, circular DNA, nicked
 circular DNA and linear DNA of same molecular weight migrate differently. Relative mobilities of these three forms
dependent primarily on the agarose concentration in gel, but also on applied current strength, ionic strength of buffer
and density of superhelical twists in the closed circular DNA. However according to various conditions in some cases the
closed circular DNA moves faster thanthe linear one which is reversed in some other cases.

Unambiguous method is to carry out electrophoresis in presence of increasing quantities of ethidium bromide. While the
concentration of ethidium bromide is increased more dye gets bound to DNA; this in turn removes the negative
superhelicity in closed circular DNA thereby decreasing the migration rate. At the critical-free dye concentration, where
no superhelicity remains, the rate of migration of circular closed DNA reaches its minimum value. When even more
ethidium bromide is added positive superhelicity is induced and mobility increases rapidly. This property can be used to
distinguish between circularly closed DNA and its other topoisomers.
Applied voltage: -
The rate of migration of DNA molecules is directly proportional to the applied voltage. Increase of voltage increases the
electric field strength and thereby increasing the mobility. Mobility of higher molecular weight DNA fragment is
comparatively increased more than the lower molecular weight ones. Thus the effective range of separation of DNA
fragments in an agarose gel decreases as the voltage increases. This is because increase in voltage causes larger DNA
fragments to migrate faster than the smaller ones.

Base composition and temperature: -

Generally the base composition of DNA and the temperature of operation does not affect the electrophoresis. The
optimal temperature can be anything between 4 - 30oC. However, gels containing less than 0.5% agarose are rather
flimsy and runs best at 4oC.

Materials required and preparation of stock solutions:Reagents required :

I X TAE buffer, Agarose, Ethidium bromide(0.5micrograms/mL), DNA solution, Gel loading buffer .

STOCK SOLUTION PREPARATION :

Tris Acetate (TAE) Buffer
STOCK SOLUTION 50x Tris Acetate EDTA (TAE) Buffer [1000ml]:
242g Tris Base
57.1ml of Glacial Acetic Acid 100ml of 0.5M EDTA (pH 8.0)

WORKING SOLUTION 1X Tris Acetate EDTA (TAE) Buffer:

40 mM Tris Acetate1 mM EDTA.

6X Gel Loading Buffer :

0.25% (w/v) Bromophenol blue0.25% (w/v) Xylene Cyanol FF 30% (v/v) Glycerol in water.

These Gel Loading Buffers serve three purposes:

They will increase the density of samples, ensuring that the DNA drops evenly into the well.
They add color to the sample, thereby simplifying the loading processes.
They contain dyes that, in electric field, move towards the anode at predictable rate.
DNA Markers for quantitative analysis of electrophoresis bands:
DNA marker working solutions are prepared from a stock provided by the manufacturer as a mixture of similar sized
fragments (e.g. 1 kb ladder or completely digested restriction products of a known genome (such as Lambda genome
digested with HindIII, /HindIII).
The working solution is made as follows:
The amount of DNA required to aliquot depends on the concentration of DNA present in the vial provided by the
manufacturer. It is required that every loading of marker in the gel contains 500ng of DNA in it. Thus for a standard
500µg/ml dye, a volume of 1µl should contain the desired amount of DNA in it.
Every time a total volume of 12µl of marker needs to be loaded into the gel. This means that for 20 reactions (say) one has
to make a total of 12x20=240µl of marker solution. The constituentsof this mix should be:
Marker: 1µl (500ng) x 20 (number of reactions)=20µl6X dye: (1/6th of 240µl)=40µl
ddH2O: 180µl
Visualizing dye - Ethidium bromide
A working solution of EtBr is made from a stock of 50mg/ml so as to make a final concentrationof 0.5µg/ml in the
TAE/TBE buffer prepared for agarose gel electrophoresis. For this, a volume of 5µl

(often 10µl to increase the intensity) is added to every 100µl of TAE or TBE. The final solution is lightsensitive.

a. 1M Tris
121.1g of Tris Base is dissolved in 800ml of water. The pH is adjusted to the desired value by
adding concentrated HCl.
pH HCl
7.4 70ml
7.5 60ml

8.0 42ml
The solution is sterilized by autoclaving (if it is yellow, then the solution is discarded)
Apparatus required:-

1. Gel electrophoresis trough with running tray and combs

2. Power supply
3. UV transilluminator
4. Micro-pipettes
5. Eppendorf tubes

PROTOCOL:
Safety note: Ethidium bromide is a carcinogen that can be absorbed through skin. Gloves must be worn
at all times when handling items that contain EtBr.

1. 1% agarose solution was made in 1X TAE buffer. Ethidium bromide was added to it to a final concentration
of 0.5 microgram/mL. This solution was heated to a clear solution in a microwave oven.
2. The solution is poured in casting tray. Comb was then inserted to make wells. The solution was allowed to
cool which results in formation of gel.
3. In a piece of butterpaper / parafilm with the help of micropipettes DNA solution and gel loading buffer
with dye is mixed. It is then loaded in the well.
4. The gel is run till the dye reaches the other end of the gel. The wells containing the DNA are placed at the
negative electrode.

5. The DNA band is observed by the UV transilluminator

Observation:

LANE 1 2 3 4 5 6 7 8

DNA I II III IV V VI VII 1kb ladder

SAMPLE
EXPERIMENT No. 7 RESTRICTION DIGESTION

AIM:
To perform restriction digestion of pcDNA3-easy vector with EcoR1 and Xho1
restriction enzymes and analyse the restriction patterns by agarose gel electrophoresis.

PRINCIPLE:
RESTRICTION ENZYME ACTIVITY:
Bacteria are under constant attack by bacteriophages. To protect themselves, many types of
bacteria have developed defence mechanisms in the form of enzymes called Endonucleases
that chop up any foreign DNA. Since these enzymes restrict the further infection of
bacteriophages, so they are termed ‘Restriction Endonucleases’.
These molecular scissors found in the bacterial cytoplasm can prove dangerous to the cell so
bacteria protect their own DNA by methylating the Adenine or Cytosine bases. The Methyl
groups block the binding of restriction enzymes but the not the normal reading and
replication of genetic information. DNA from an attacking bacteriophage will not have these
protective methyl groups and will be destroyed.
Together Restriction enzymes and its modification Methyltransferase form a Restriction
Modification System.
The Type II restriction enzyme is mostly used and they recognise specific DNA sequences and
cleave the DNA at fixed locations at/near the recognition sites. They act as a dimer: each
subunit recognizing the same 5’ to 3’ nucleotide sequence in complementary DNA strand and
hence are said to recognize pallindromic sequences.
EcoR1 recognises the following sequence and cleaves each backbone between G and A base
residue giving 5’ protruding ends.

5’- GAATTC-3’ 5’-G AATTC-3’

3’-CTTAAG-5’ 3’-CTTAA G-5'

Xho1 recognises the following sequence and cleaves each backbone between C and T base
residues giving 5’ protruding ends. Xho1 is obtained from Xanthomonas holcicola.

5’-CTCGAG-3’ 5’-C TCGAG-3’

3’-GAGCTC-5’ 3’-GAGCT C-5’

Restriction enzymes are naturally occurring endonucleases that recognize a specific

palindromic sequence (4-12 bp) and cut the double strand phosphate backbone of DNA within
or near the recognition sequence. The cuts may be opposite each other on the DNA backbone
or staggered. Opposite cuts produce blunt ends, while staggered cuts produce sticky ends.
For example, EcoRI recognizes the palindrome sequence, GAATTC (note the reverse
complement of this sequence is GAATTC), and cleaves the DNA 's phosphate backbone
between the G and the A on both strands generating staggered ends that are complementary
to each other:
Because restriction endonucleases recognize specific sequences and cut DNA to produce
sticky ends, they are useful for gene splicing manipulations.Another enzyme EcoRV
creates blunt ends:

Both sticky and blunt ends may be rejoined by another DNA modifying enzyme DNA ligase.
Restriction enzymes are named after the organism they are isolated from and usually the
temporal order of their isolation. For example EcoRV is the fifth reported from E. coli.
Restriction enzyme digestions are performed by incubating double-stranded DNA
molecules with an appropriate amount of restriction enzyme, in its respective buffer as
recommended by the supplier, and at the optimal temperature for that specific enzyme. The
optimal sodium chloride concentration in the reaction varies for different enzymes, and a set
of three standard buffers containing three concentrations of sodium chloride are prepared and
used when necessary. Typical digestions included a unit of enzyme per microgram of starting
DNA, and one enzyme unit usually (depending on the supplier) is defined as the amount of
enzyme needed to completely digest one microgram of double-stranded DNA in one hour at
the appropriate temperature. These reactions usually are incubated for 1-3 hours, to insure
complete digestion, at the optimal temperature for enzyme activity, typically 37deg C.

FACTORS AFFECTING RESTRICTION ENZYME ACTIVITY:

1. TEMPERATURE:
Most digestions are carried out at 370C. However there are a few exceptions like
XmaI is carried out at lower temperatures (250 C) while Taq1 at higher
temperature (650C).

2. BUFFER SYSTEM:
Tris HCl is most commonly used buffering agent in incubation mixtures which is
temperature dependent. Mostly, restriction enzymes are active in pH range 7-8.

3. IONIC CONDITION:
Mg2+ is an absolute requirement for all restriction endonucleases but the
requirement of other ions (Na+ and K+) varies with different enzymes.

4. METHYLATION of DNA:
Methylation of specific Adenine or Cytidine residues within the recognition
sequence of the Restriction Enzymes affects the digestion of DNA.

In our experiment, we used PIN-1 cDNA which was isolated by reverse transcription of
total RNA of C. elegans using PIN-1 specific gene primers
and subcloned into pcDNA3 Easy. This gene has a size of 500
bp. The insert was amplified such that the recognition sites
for the restriction enzymes EcoRI and XhoI were generated
on its ends. So, in pcDNA3 Easy, the insert has the restriction
enzyme sites EcoRI and XhoI on its either side. The vector
itself has two recognition sites for EcoRI on either site of the
insert cloning site. So, in effect, there are three recognition
sites for activity of EcoRI and one site for XhoI in the insert
ligated vector.
Materials required:

1. 10X assay buffer

2. 2.5X Gel Loading Buffer
3. pGEMT-easy vector (template for Restriction enymes)
4. Xho1 (Bangalore GeneI)
5. EcoR1(Bangalore GeneI)
6. 1X Bovine Serum Albumin
7. 6X gel loading dye
8. Agarose (for electrophoresis)
9. 1X TAE (for electrophoresis)
10. 1 Kb ladder
11. Distilled Water

Equipments and glassware:

Beakers, Conical flasks, measuring cylinder, micropipettes, tips, electrophoretic
apparatus.

Procedure:
Setting up the Restriction Digestion reaction:
1. The vials containing the Restriction Enzymes were kept in ice from -20
degrees in order to thaw them.
2. Two different reaction mixes were prepared according to the following
constituents:

EcoRI Reaction Mix:

S. Constituents Volume (in µl) for One Volume (in µl) for 10
No. Reaction Reactions (X 11)
1 Template DNA 6 66
2 10X Buffer 2 22
3 EcoRI enzyme 1 11
4 ddH2O 9 99
5 BSA 2 22
6 TOTAL 20 220

XhoI Reaction Mix:

 S. Constituents Volume (in µl) for One Volume (in µl) for 4
No. Reaction Reactions (X 4.5)
1 Template DNA 6 27
2 10X Buffer 2 9
3 XhoI enzyme 1 4.5
4 ddH2O 9 40.5
5 BSA 2 9
6 TOTAL 20 90

3. The mix was prepared on ice and the constituents were mixed well and
incubated at 370C overnight.
4. The restriction digestion reactions were mixed with 6 µl of gel loading dye
and loaded into the wells on the gel.
5. The samples were run on gel for 1 hour at 100 V.
6. The results were checked in the gel documentation machine.

Observations.

DNA Samples Used:

Lane 1 : Sample 2 cut by E.coli
Lane 2 : Sample 5 cut by E.coli
Lane 3 : Sample 2 cut by xho 1
Lane 4 : Sample 5 cut by xho 1
Lane 5: 1 kb ladder (in the left image)

1 2 3 4 1 2 3 4 5
Experiment No.8 PCR
AIM.
•To perform PCR amplification of specific target sequence from genomic DNA.
•To analyze the amplified product by agarose gel electrophoresis.

PRINCIPLE:
The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene.
The cycling reactions: There are three major steps in a PCR, which are repeated for 30 or 40
cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction
mixture in a very short time.

1. Denaturation at 94°C: During the denaturation, the double strand melts open to single
stranded DNA, all enzymatic reactions stop (for example: the extension from a previous
cycle).

2. Annealing at 52°C ~58°C: The primers are jiggling around, caused by the Brownian motion.
Ionic bonds are constantly formed and broken between the single stranded primer and the
single stranded template. The more stable bonds last a little bit longer (primers that fit
exactly) and on that little piece of double stranded DNA (template and primer), the
polymerase can attach and starts copying the template. Once there are a few bases built in,
the ionic bond is so strong between the template and the primer that it does not break
anymore.

3. Extension at 72°C: This is the ideal working temperature for the polymerase. The primers,
where there are a few bases built in, already have a stronger ionic attraction to the template
than the forces breaking these attractions. Primers that are on positions with no exact match
get loose again (because of the higher temperature) and don't give an extension of the
fragment.

The bases (complementary to the template) are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added
complementary to the template). Because both strands are copied during PCR, there is an
exponential increase of the number of copies of the gene. Suppose there is only one copy of the
wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles,
there will be 4 copies, three cycles will result in 8 copies and so on.
MATERIALS REQUIRED:
DNA Template: DNA purified from Serratia marcescens.
Primers: Two primers i.e. forward primer & reverse primer

Materials

10X Assay Buffer

10 mM dNTP Mix
100 bp DNA ladder
1 kb DNA ladder
Forward Primer

Reverse Primer
Nuclease Free Water
Template DNA
Taq DNA Polymerase

Gel Loading Buffer

Agarose
50XTAE

PCR Tubes

Other: Thermocycler, Ethidium Bromide, UV transilluminator, Tips, Gloves, Micropipette etc

MgCl2 (already contained in buffer, 15 mM; final concentration 1.5 mM)

PCR is very sensitive to contamination from outside DNAs. Steps should be taken to reduce the
chance for contamination, such as wearing gloves, using aerosol tips (tips with a wad of cotton at
the top). Something that IS important is to assemble your reactions on ice.

The final concentrations of reagents in PCR reactions are as follows:

Buffer: 1X, usually comes as 10X stock. For 50µL reactions, this means 5µL.

dNTPs: for most general PCR, you want the final concentration to be 200µM, so a 2mM stock is
essentially 10X -- For 50µL reactions, use 5µL.

Primers: a good place to start with primer concentration is 20~50pmol of each primer per
reaction. It may be increased to 75pmol or 100pmol the desired product is not obtained.

Template: We can get product with incredibly small amounts of starting DNA.

MgCl2: 1X, this is the greatest variable in PCR. The success of a PCR is very dependent on how
much magnesium is present in the reaction. For this reason, it is usually advisable to do a
magnesium optimization when performing new PCRs.
Procedure:
Setting up for PCR:

S. No. Constituent Volume for 1 Reaction Volume for 5

(in microliters) Reactions
(1 reaction extra)
(in microliters)
1 Sterile DDW 38 190
2 10 X Buffer (containing 15 mM 5 25
MgCl2)
3 dNTP Mix (containing 200μM conc.) 3 15
4 Forward Primer 1 5
5 Reverse Primer 1 5
6 Taq Polymerase 1 5
7 Template DNA 1 5
TOTAL 50 250

• All the components except (Taq pol & template DNA) was mixed gently.
• Taq pol & template DNA were added in PCR tubes directly 1 µl each after addition
of 48 µl from the mixture.

PCR amplification:
30 cycles were carried out the amplification in a thermocycler using the following reaction conditions:

Initial denaturation Denaturation Annealing Extension

94 ֯ C 94 ֯ C 48 ֯ C 72֯C
1min 30sec 30sec 1min

Analysis on Agarose gel:

• Following the PCR amplification,10 µl 6X loading dye was added in 50 µl of PCR product in
PCR tubes.
• Tapped the mixture thoroughly for mixing.
• 40 µl from each PCR tubes were loaded in the well of the 1.5% agarose gel(Ethidium
bromide was added at a concentration of 0.5 µg/ml)
• 100bp & 1kb ladder were added.
• the samples were electrophoresed at 100 volts for 1-2 hours till the tracking dye
(bromophenol blue) reaches 314th of the length of the gel.
• Visualized the gel under a UV transilluminator.
Observations

Gel Loading Order

Lane: 1 2 3 4 5 6
Sample no 100bp Tube 1 tube2 tube3 tube4 1kb ladder
ladder

1 2 3 4 5 6

Interpretation:
As observed on agarose gel, PCR amplification of the template using specific primers results in a
specific product of a particular length. The conditions have been optimized to give a highly specific
product of ~750bp as is observed by the absence of any non-specific products. Altering these
conditions would result in non-specific amplification.