UB201

Molecular Biology Lab Reports

Ankur Bhaumik
18895

Lab-1 (Performed Online)

Competent Cell Preparation and Transformation

Rationale- Plasmids are very important vectors that can be used to introduce a
certain gene into a cell. This can then be used to study the function of the gene or
to synthesize proteins for other application. However, for the cell to express the
gene it is necessary for the plasmid DNA to be present inside the cell. Making the
cell competent means making it ready to accept the plasmid DNA. Ones the
plasmid DNA has been taken up by a cell, it is transformed as it has more genetic
material than it previously had. It is also necessary to separate the transformed
cells from non-transformed ones.

Materials Required-
A) Competent cell preparation
1. Laminar Hood 7. E. Coli TG1 culture in LB medium
2. Ice at 4˚C 8. Magnesium chloride (0.1 M)
3. Centrifuge Machine 9. Calcium chloride (0.1 M in 15%
4. Microfuge Tubes glycerol)
5. Vortex Machine 10. Liquid Nitrogen
6. Fridge

B) Transformation
1. Laminar Hood 5. Incubator
2. Ice at 4˚C 6. 100 ng Plasmid DNA (PBad His B)
3. Centrifuge Machine 7. LB-Ampicillin plate
4. Water bath 42 ˚C 8. LB stock solution
Protocol

A) Competent Cell Preparation

Subculture an overnight grown bacteria as 1:100 in LB medium.

Incubate at 37 degree celsius while shaking at 180 rpm till it reaches an OD of

0.5-0.6 (at 595 nm).

Pipette 2 mL of the culture and chill it on ice for 5 mins

Spin at 7000 rpm for 10 mins at 4 degree celsius. Discard the supernatant and
other debris without disturbing the bacterial pellet

Resuspend the pellet in 200 L of ice cold 0.1 M solution and keep it
on ice for 30 mins.

Spin at 7000 rpm for 10 mins at 4 degree celsius

Discard supernatant and other debris without damaging the pellet.

Resuspend it in 50 of 0.1 M CaCl2 with 15% glycerol.

Freeze and store the now “competent cells” at -80 degree

celsius
B) Transformation
Thaw it on ice and then add 3 L of plasmid DNA. Mix by flicking with finger

Keep on ice for 20 mins

Give heat shock for 45s and 90s at 42 degree celsius and then immediately snap chill

Add 1 mL of LB and incubate at 37 degree celsius for 45 mins

Centrifuge the cell mixture at room temperature at 10000 rpm for 1 minute

Discard supernatant and leave around 50 L of medium in tube

Resuspend the bacteria in the remaining media and then plate them

Incubate overnight for 37 degree celsius.

Count the number of colonies and then calculate the transformation efficiency.
Transformation efficiency = [(cfu on plate)/ (ng of DNA)]*( 1000 ng / g)*(final dilution)
Results

Observation

1. All the four plates on the left show a large number of colonies (>300) for both
durations.
2. No colonies are observed LB+ Ampicillin + Competent cells
3. In case of LB+ Ampicillin+ Competent cells + Plasmid 277 colonies are counted
for 45 s heat shock and 158 colonies for 90s heat shock. There are no satellite
colonies.

Calculations

45s
277
𝑇𝑟𝑎𝑛𝑠𝑓𝑜𝑟𝑚𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = × 1000 × 1 𝑐𝑓𝑢/𝜇𝑔 𝐷𝑁𝐴 = 2770 𝑐𝑓𝑢/𝜇𝑔 𝐷𝑁𝐴
100

90s
158
𝑇𝑟𝑎𝑛𝑠𝑓𝑜𝑟𝑚𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = × 1000 × 1 𝑐𝑓𝑢/𝜇𝑔 𝐷𝑁𝐴 = 1580 𝑐𝑓𝑢/𝜇𝑔 𝐷𝑁𝐴
100
Inference/ Discussion
1. The four plates on the left had a large number of colonies because there was
no Ampicillin in the medium and the cells were able to grow freely.
2. No colonies are observed LB+ Ampicillin + Competent cells because they are
not resistant to antibiotics.
3. In case of LB+ Ampicillin+ Competent cells a moderate number of colonies
are observed. These are colonies of the transformed cells. They are resistant
to ampicillin and are thus able to grow. The parents of these cells were able
to take up the vector plasmids.
4. A larger number of colonies are observed for 45s heat shock than 90s
because a longer heat shock might kill the cells. Thus, for transformation
there is an optimal heat shock duration at which transformation efficiency
will be maximum. Beyond this the transformation efficiency decreases.
 Lab 2 (Performed Offline)
Plasmid Isolation and Restriction Enzyme

Rationale- To use plasmids as vector we need to first isolate them so that the gene
of interest can be ligated onto it. We need to separate the plasmid DNA from all
other molecules in the cell. This is achieved by using alkaline lysis in which the cell
membrane, proteins and chromosomal DNA is denatured and then separated
using differential precipitation.

Materials Required- (Offline Protocol)

I. Extraction of plasmid
1. Centrifuge
2. Vortex Machine
3. Pipettes
4. Buffers-P1, P2, N3, PB, PE, EB
5. QIAprep 2.0 Spin Column

II. Quantification using nanodrop

1. Mili Q water
2. Nanodrop

III. Qualitative analysis using gel electrophoresis

1. Agarose gel with wells
2. TAE buffer
3. Loading Dye
4. Ethidium Bromide
Protocol
I. Extraction of plasmid
Transfer 1.25 ml of culture to a sterile 1.5 ml tube. Harvest the bacterial cells by
centrifugation at 10000 rpm for 3 min at 40C.

Remove the supernatant and resuspend the pelleted bacterial cells in 250 μl Buffer P1 and transfer the
suspension to the second tube and resuspend the bacterial pellet

Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube.

Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times

Centrifuge for 10 min at 40C, 13,000 rpm

Apply 800 μl of the supernatant from previous step to the QIAprep 2.0 Spin Column.
Centrifuge for 50 s at 13,000 rpm. Discard the flow through

Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and
centrifuging for 50 s at 13,000 rpm . Discard the flow through

Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s at 13,000
rpm

Discard the flow through, and centrifuge at full speed for an additional 1 min at 13,000
rpm

Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer
EB to the center of each QIAprep 2.0 Spin Column, let stand for 1-2 min, and centrifuge for 1 min at 13,000
rpm
 II. Quantification using nanodrop
Clean the sample port with M.Q water

Select the parameters for DNA quantification

Blank with Elution Buffer

Measure your sample and determine 260/230 & 260/280 ratios

III. Qualitative analysis using gel electrophoresis

Make agarose gel with wells. The gel must contain EtBr

Add 2 µl ‘loading buffer’ to 10 ul of the plasmid soln (elute)

Each sample is ‘loaded’ into a well in the agarose gel.

The gel electrophoresis apparatus is set up as explained by the instructor

The ‘running buffer’ used is 1X TAE

The gel is ‘run’ at 80-100 V for 30-60min

After the run is complete, plasmid DNA bands are visualized using a Gel
doc apparatus

Results-
Gel Electrophoresis-

Nanodrop-

Concentration of isolated plasmid DNA = 49.6 ng/μl

260/280 ratio = 1.83
260/230 ratio =1.86

Inference-
1. The electrophoresis results clearly show the linear and supercoiled forms of
plasmid DNA. All the three forms are visible in the control sample.
2. The smudge like feature in the image is mostly denatured RNA.
3. Concentration of isolated plasmid DNA is 49.6 ng/μl.
4. 260/280 ratio is 1.83 which shows that contamination by proteins is negligible
(Ideal Value 1.8-2)
5. 260/230 ratio is 1.86 shows that there is some contamination by organic
compounds and chaotropic salts (Ideal value is 2-2.2).
 Lab 3 (Online)
Testing the UV Sensitivity of UvrB Negative E. Coli

Rationale-UV radiation leads to the formation of thymidine dimer. This dimer is

unable to fit into the DNA helix and hence it hinders the normal replication and
gene expression processes and thus it needs to be repaired. There are two
mechanisms for DNA repair Photoreactivation and Nucleotide Excision Repair
(NER). Various proteins such as UvrA, UvrB, UvrC, UvrD take part in NER. What we
intend to find is if UvrB is an important ingredient of this mechanism. This is
achieved by using gene-knockoff method, where the gene of interest is removed
and then the same process is observed.

Materials Required
1. Pipettes
2. Spreader
3. Microfuge tubes
4. Spectrophotometer
5. Temperature controlled incubator
6. Centrifuge
7. LB plates
8. LB broth
9. Two strains of E. Coli - TG1 and TG1 UvrB
Protocol
Overnight grown bacteria cultures (TG1 and TG1 UvrB) is subcultured as 1:100
in LB medium

Then we incubate this at 37 degree celsius while rotating at 180 rpm till the OD reaches 0.5.

10 fold serial dilutions were made up to 10-5. 900 l of LB broth is added to all the five tubes
corresponding to the dilutions. They are labelled TG1-1, TG1-2, TG1-3, TG1-4 and TG-5.

Prepare five dilutions for the UvrB strain

Take four LB plates and then labelled them according to the time they are going to be exposed to
UV. (0s, 5s, 10s, 15s)

Mark 8 spots on each LB plate such that we have 2 spots for each concentration of both wild and
mutant type. We consider only the concentrations 10-1, 10-2, 10-3, 10-4

Before pipetting vortex the tubes and then pipette 5 μl of the culture and drop it at the
designated spots. After this dry the spots

Then the plates are exposed to UV as decided (the plates are kept open during exposure).
After this incubate it at 37 degrees overnight and observe the plates the next day
Results

Inference
1. At no UV exposure both the strains grow to almost an equal extent.
2. As expected for both strains the density of colonies decreases as the the
dilution is increased.
3. The wild strain shows much less reduction in the sizes of colonies as
compared to the mutant strain as the exposure time is increased
4. For the mutant strain, for higher dilutions the colonies almost disappear even
when the exposure time is only 5s. When exposure time is 15s, the colonies
disappear also for lower dilutions.
5. At a particular dilution and at a particular exposure time(non-zero), the wild
strain grows better than the mutant strain. This proves that UvrB is essential
for DNA repair using NER.
6. At higher exposure time and higher dilution even, the wild strain shows
reduction in the size of the colony. This is because in dilute solutions more
DNA damage is being done by UV as bacterial cells are not shielded by other
bacterial cells.
 Lab 4 (Online)
β-Galactosidase Assay and Plaque Assay

A. β-Galactosidase Assay

Rationale- β-galactosidase is a protein encoded the lacZ gene of the lac operon in
E. coli. It can hydrolyse -D- galactosides. The compound o-nitrophenyl- D-
galactoside (ONPG) is also recognised as a substrate and cleaved to yield galactose
and o-nitrophenol that gives a yellow colour. Thus, we can use this colour
generation due to o-nitrophenol production to quantify -galactoside
concentration.

Materials Required
a. Conical flasks
b. Falcon tubes
c. Microfuge tubes
d. Heat blocks
e. Pipettes
f. Vortex machine
g. Spectrophotometer
h. Plate reader
i. LB Broth (0.5X)
j. Isopropyl β- d-1-thiogalactopyranoside (IPTG)
k. Chloroform
l. Sodium dodecyl sulphate (SDS)
m. O-nitrophenyl-β-D-galactoside (ONPG)
n. Sodium carbonate
o. Magnesium sulphate
p. Potassium chloride
q. Monosodium phosphate
r. Disodium phosphate
s. β-Mercaptoethanol (β-ME)
t. Milli-Q water
 u. LacZ buffer

Protocol-
A subculture of bacteria was prepared in 0.5X (8ml) of LB and grown till OD of the
culture was 0.45-0.55 at 37 degree Celsius and 600nm

The culture was induced with 1mM of IPTG and incubated at 37 degrees for 7
minutes.

Then the culture was kept in ice for 15-30 minutes

500 μl of the culture was aliquot into a mix of 300 μl of

LacZ buffer + 40 μl of chloroform + 20 μl of 0.1% SDS

Then the sample was vortexed for 15-30 seconds and kept on ice for 30
mins

It was again vortexed and kept at 30 degrees to equilibrate for 15 minutes.

200 µL of 12 mg/mL ONPG substrate was added, mixed, vortexed, and kept at 30
degrees

After an hour the reaction is stopped by adding 500 μl of

1M sodium carbonate and vortexing.

The samples are then centrifuged at 13000 rpm for 15 minutes at roomtemperature.
Then we took the supernatant and then dilute it 2-fold usingMilli-Q water

Measured OD at 420nm and 550nm. Calculated activity in miller

units
Results-
Sample Absorbance at 420 nm Miller units
IPTG induced A 0.2236 28.945
IPTG induced B 0.2536 32.828
IPTG induced C 0.2308 29.877
Uninduced A 0.1683 21.786
Uninduced B 0.1673 21.657
Uninduced C 0.1621 20.984

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 420 𝑛𝑚 𝑓𝑜𝑟 𝐼𝑃𝑇𝐺 𝑖𝑛𝑑𝑢𝑐𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 0.236 𝑜𝑟 30.55 𝑀𝑈

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 420 𝑛𝑚 𝑓𝑜𝑟 𝑢𝑛𝑖𝑛𝑑𝑢𝑐𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 0.1659 𝑜𝑟 21.475 𝑀𝑈

Inference-
1. O-nitrophenol is formed as confirmed by the slight yellow colour in the
induced samples.
2. Absorbance at 420 nm is much greater for the induced samples as compared
to the uninduced samples. This confirms that IPTG induces the expression of
lacZ gene. This is because more o-nitrophenol after a fixed time means more
β-galactosidase produced during 7 min incubation as ONPG is in excess.
More β-galactosidase means greater expression of lacZ gene.
3. Uninduced samples also have significant absorbance at this wavelength
because a small level of expression of lacZ gene exists even in the uninduced
sample.
B. Plaque Assay

Rationale- Bacteriophages are viruses that infect bacteria. They use bacterial
machinery to replicate themselves. Lytic viruses kill their host cells after infecting
them. These host cells then release several new virus particles which infect other
nearby bacterial cells. Thus, these appear as plaques on the bacterial lawn. Plaque
assay uses the number of plaques formed to quantify the infecting ability of a viral
culture. It is also an indirect indicator of the total no. of viral particles in the
culture.

Materials Required

a. Water bath
b. Test tube rack
c. Petri plates
d. Pipettes
e. Thermometer
f. Burner
g. Incubator
h. Micro-centrifuge tubes
i. Culture of Escherichia coli KL16
j. P1 phage stock
k. 1.8 % LB agar
l. 0.8% LB soft agar
Protocol-

Prepare 10-fold dilution of the phage stock culture from 10-1 to 10-10

Take five tubes of LB soft agar and five LB agar Petri plates labelled as 10-5 through 10-10.

Place the five labelled soft LB agar tubes (5 mL) into a water bath. Wateris kept
such that the depth is just slightly above that of the agar in the tubes. The agar is
melted at 100 ̊C and then the melted agar is maintained at 45 ̊C.

0.1 mL of overnight bacterial Escherichia coli KL16 culture was

added to the LB soft agar and mixed gently

Add 0.1 ml of each serial dilution of phage to its corresponding soft agar tube, mixed properly

Then we poured the soft agar mix on to the hard LB agar Petri plate with the
corresponding label, let the soft agar solidify and incubate all plate cultures in an
inverted position at 37 ̊C for 8-24 h.

The titer of the viral stock is

calculated
Results-

i) The plate with 10-5 dilution had too many plaques ( > 300) and hence the
number of plaques is not counted.
ii) The plate with 10-6 dilution had 124 plaques.
iii) The plate with 10-7 dilution had 79 plaques.
iv) The plate with 10-8 dilution had 6 plaques.
v) The plate with 10-9 dilution did not have any plaques. (Picture not shown)

Calculation of titer

𝑃𝑙𝑎𝑞𝑢𝑒 𝑓𝑜𝑟𝑚𝑖𝑛𝑔 𝑢𝑛𝑖𝑡(𝑝𝑓𝑢/𝑚𝑙 ) = (𝑛𝑢𝑚𝑏𝑒𝑟𝑜𝑓𝑝𝑙𝑎𝑞𝑢𝑒𝑠)/(𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 × 𝑣𝑜𝑙𝑢𝑚𝑒𝑜𝑓𝑑𝑖𝑙𝑢𝑡𝑒𝑑𝑣𝑖𝑟𝑢𝑠)

𝑉𝑜𝑙𝑢𝑚𝑒 𝑡𝑎𝑘𝑒𝑛 𝑓𝑜𝑟 𝑝𝑙𝑎𝑡𝑖𝑛𝑔 = 0.1 𝑚𝑙
 Dilution No. of plaques pfu/ml
10-5 >300 -
-6
10 124 1.24 X 109
10-7 79 7.9 X 109
10-8 6 6 X 109
10-9 0 0

Since, no. of plaques should be between 30 and 300 we take the average of 10-6
and 10-7 dilutions. Thus, the viral culture has an average of 4.57 X 109 pfu/ml.

Inference-
1) The number of plaques is very high at 10-5 dilution. It becomes impossible to
distinguish one plaque form another as in many cases the plaques have
merged.
2) At 10-9 dilution it is impossible to detect plaques with naked eye.
3) The given sample has an average titer of 4.57 X 109 pfu/ml.