Slide 1: Title Slide
- Genomic Library Construction: The Role of Restriction Enzymes and Transformation Success

Slide 2: Objective and Process Overview
- Construct a genomic library using E.coli Top10 genomic DNA and pUC19 plasmid DNA
- Confer streptomycin resistance to a recipient E.coli strain Mach1 through the functional selection of the rspL gene
- Briefly mention the steps: Gel electrophoresis, ligation reaction, transformation

Slide 3: Gel Electrophoresis and Restriction Enzymes
- Summarize initial gel electrophoresis error due to low genomic DNA concentration
- Mention the use of cleaned-up genomic DNA and the addition of HindIII enzyme
- Show Image 6 as an example of successful digestion

Slide 4: Troubleshooting Transformation
- Show Image 5 (unsuccessful transformation)
- Quickly mention possible issues with competent cells preparation, ligation reaction, and transformation protocol
- Suggest potential improvements and address human errors

Slide 5: Future Directions and Conclusion
- Explore alternative restriction enzymes, plasmid vectors, and competent cell strains
- Ensure proper recovery time, nutrient-rich medium, and appropriate plating methods
- Include positive and negative controls in all experiments
- Wrap up the presentation and emphasize the significance of troubleshooting and optimization


according to the above slides, write me a presentation script. You can use the information from below:

Results
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Image 1: Picture of first 1% gel electrophoresis performed to confirm the presence of rspL gene
after digestion of genomic DNA with BamHI
The above image was captured from UV light analysis of the 1% agarose gel prepared in 1X TAE
buffer with 2.5 µL INtRON RedSafeTM and 6X loading dye. The well labelled 1 was utilized for
1Kb plus DNA ladder RTU (FroggoBI0- BIOHELIX), 5 µL loaded as standard whilst the well
labelled 2 contains the digested genomic DNA (10 µL digested sample + 2 µL loading dye). The
above gel electrophoresis was run at 120 V for 20 minutes. Genomic uncut was not loaded
which was considered an error. Moreover, there were no bands at all and the problem was low
concentration of genomic DNA.
It was noticed that since INtRON RedSafeTM requires at minimum 50 ng/ µL of the sample
concentration to give any visualization detection effects, the above gel electrophoresis was
unsuccessful. This is because the concentration of originally isolated genomic Top10 DNA was
already quite low with 33.2 ng/ µL and while preparing the digestion reaction with total volume
of 35 µL we used 10 µL of the genomic DNA which implies that our genomic was diluted. Not
only this when we loaded 10 µL digested sample with 2 µL loading dye it further diluted. As per
this, the concentration of loaded sample was 33.2 ng/ µL which is very less than 50 ng/ µL as
per the RedSafeTM to work efficiently.
Calculations of digested sample concentration:
(33.2 ng / µL × 10 µL in digestion) / (10 µL while loading) = 33.2 ng/ µL

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Hence, nothing was detected.Furthermore, digested pUC19 plasmid and uncut genomic Top10
was not loaded and therefore nothing was there to compare which was a mistake.
Image 2: Second 1% gel electrophoresis performed after digesting newly cleaned genomic DNA.
The above image represents the picture captured of 1% gel electrophoresis run for 25 minutes
at 120 V under UV light. This is the seconds gel that contained new digestion reaction
containing the genomic DNA after clean up. The gel was prepared in 1X TAE buffer with 2.5 µL
INtRON RedSafeTM along with 6X loading dye used for sample preparations. Gel was shared with
another group (Mahan’s) which is represented with the arrow head and samples in lane 1,2,3
belongs to other group. Well labelled 4 contained the 5 µL of 1Kb plus DNA ladder RTU
(FroggoBI0- BIOHELIX) , well 5 contained the uncut genomic Top10 (5 µL + 6X loading dye), well
6 contained cut genomic with BamHI((5 µL + 6X loading dye) and well 7 contains the cut
plasmid with BamHI ((5 µL + 6X loading dye).
Image 3: BIORAD image of 1% gel electrophoresis performed for confirming the action of
BamHI on rspL gene of genomic DNA and on plasmid pUC19.
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The above image represents the picture captured of 1% gel electrophoresis run for 25 minutes
at 120 V under BIORAD Imager. The gel was prepared in 1X TAE buffer with 2.5 µL INtRON
RedSafeTM along with 6X loading dye used for sample preparations. Gel was shared with
another group (Mahan’s) which is represented with the arrow head and samples in lane 1,2,3
belongs to other group. Well labelled 4 contained the 5 µL of 1Kb plus DNA ladder RTU
(FroggoBI0- BIOHELIX) , well 5 contained the uncut genomic Top10 (5 µL + 6X loading dye)
which as expected showed a large uncut band, well 6 contained cut genomic with BamHI((5 µL
+ 6X loading dye) which showed a large smear in image and is shorter as compared to the large
band of genomic in lane 5 hence suggest the digestion to be complete and well 7 contains the
cut plasmid with BamHI ((5 µL + 6X loading dye) which showed two very faint bands as
highlighted with red color on image.
Image 4: Transformation results containing experiment results and controls after 24-hour
incubation at 37 degrees Celsius.
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The above image represents the picture captured of agar plates after 24-hour incubation at 37
degrees Celsius. Each of the plates above contains 100 µL of 25 mg/mL Ampicillin, 40 µL of 20
mg/mL X-gal, and 8 µL of 500 mM IPTG. Negative control (C) contains 50 µL of competent cells.
Positive control (A) contains 50 µL of competent cells and 5 µL of pUC19. Experiment plates are
in duplicate (B , D) and contains 50 µL of competent cells and 10 µL of ligated product. The
experiment failed since the positive control contains contamination (the big white colonies
shown in the image are contamination).
Image 5: Transformation results containing experiment results and controls after 24-hour
incubation at 37 degrees Celsius. Experiment was performed due to failure of the first one.
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The above image represents the picture captured of agar plates after 24-hour incubation at 37
degrees Celsius. Each of the plates above contains 100 µL of 25 mg/mL Ampicillin, 40 µL of 20
mg/mL X-gal, and 8 µL of 500 mM IPTG. Negative control (D) ,Positive control (C) ,experiment
plates (A,B) in duplicate contain the contents as mentioned in Image 4 above The experiment
was not successful due to plating issue. There is inefficient uptake of DNA by host organism
which led to inefficient transformation that mean unsuccessful transformation.
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Image 6: Third 1% gel electrophoresis performed using HindIII and BamHI.
The above image represents the picture captured of 1% gel electrophoresis run for around 45
minutes at 120 V under UV light. This is the third gel that contained a new digestion reaction
containing the genomic DNA. The gel was prepared in 1X TAE buffer with 2.5 µL INtRON
RedSafeTM along with 6X loading dye used for sample preparations. Well labelled 1 contained
the 10 µL of 1Kb plus DNA ladder RTU (FroggoBI0- BIOHELIX), well labelled 2 contained the
undigested genomic Top10 (10 µL + 6X loading dye), well labelled 3 contained digested genomic
with BamHI and HindIII (10 µL + 6X loading dye), well labelled 4 contains the undigested
plasmid (10 µL + 6X loading dye) and well labelled 5 contains the digested plasmid with BamHI
and HindIII (10 µL + 6X loading dye).
Image 7: Information regarding the total length of the genomic Top 10 DNA using restriction
analyzer
1 2 3 4 5 1 kb DNA ladder Undigested genomic Digested genomic Undigested pUC19 Digested pUC19
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The above image provides information related to the total length of the genomic Top 10 DNA
that is 4537 bp. The tool used for this was restriction analyzer. It also shows that BamHI cuts at
2 different sites on genomic DNA. The cutting positions are 180 and 2865. These sites give very
big fragments that can be seen as smear in image 2.
This enzyme cuts at 662 on pUC19 and creates sticky ends. The total size of the plasmid is 2686
bp. If we incorporate this plasmid inside a host mentioned above, it gives a smear. There are
also chances that host could not uptake the DNA containing rspL gene.
Image 8: Information regarding the total length of the genomic Top 10 DNA and cutting
positions using HindIII and BamHI enzymes using restriction analyzer.
The above image provides information related to the total length of the genomic Top 10 DNA
that is 4537 bp. The tool used for this was restriction analyzer. It also shows that BamHI cuts at
2 different sites on genomic DNA and HindIII cuts at 4130 site on genomic DNA. The cutting
positions for BamHI are 180 and 2865 and for HindIII is 4130. We used HindIII to make sure that
our fragment contains rspL gene.
Image 9: Information regarding the total length of the plasmid DNA using restriction analyzer
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The above image provides information related to the pUC19 plasmid DNA that is 2686 bp long.
The tool used for this was restriction analyzer. It also shows that BamHI cuts at 662 position on
plasmid DNA and HindIII cuts at 632 positions. Since we are using two different enzymes, there
is a better chance that our fragment contains rspL gene.
Discussion
The primary objective of this project was to create a genomic library using a streptomycinresistant strain of E.coli Top10 (4537 bp) as the genomic DNA, incorporating it into a pUC19
plasmid (2686 bp) vector (Cirino et al., 2003). The successful construction of this genomic
library was expected to confer streptomycin resistance to a recipient E.coli strain Mach1
through the functional selection of the rspL gene (Hanahan, 1983). However, throughout the
experiment, several challenges were encountered, leading to changes in the experimental
design and troubleshooting of errors.
An initial gel electrophoresis performed to confirm the presence of the rspL gene after digestion
of genomic DNA with BamHI resulted in no visible bands. This was primarily due to the low
concentration of the originally isolated genomic Top10 DNA (33.2 ng/µL), which was further
diluted during the digestion reaction and loading of the gel (Inoue et al., 1990). This
concentration was below the minimum detection limit required for the INtRON RedSafeTM
nucleic acid stain to work efficiently (50 ng/µL). As a consequence, no bands were detected,
and the experiment had to be repeated with a new and cleaned up genomic DNA sample.
Subsequent gel electrophoresis performed after digesting the cleaned-up genomic DNA with
BamHI showed successful digestion of the genomic Top10 DNA, as evidenced by a shorter
smear in comparison to the uncut band of the genomic DNA (Sorek et al., 2013). In addition, the
plasmid pUC19 showed two very faint bands after digestion with BamHI, indicating appropriate
digestion of the plasmid as well.
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A ligation reaction was set up using a 1:1 ratio for cut genomic Top10 DNA (insert) and cut
plasmid pUC19 (vector) (Chen & Dubnau, 2004). After overnight incubation at 4 °C, the ligated
product was transformed into Mach1 chemically competent cells. The initial transformation
results were inconclusive due to contamination observed in the positive control plate.
Moreover, the second transformation attempt resulted in an unsuccessful transformation due
to inefficient uptake of DNA by the host organism, which might have been caused by several
factors, such as improper preparation of competent cells, issues concerning the ligation
reaction, or the transformation protocol itself (Inoue et al., 1990).
To increase the likelihood of obtaining a fragment containing the rspL gene, a third gel
electrophoresis was performed using HindIII in addition to BamHI for digesting both genomic
Top10 DNA and plasmid pUC19 (Cirino et al., 2003). The use of two different enzymes would
increase the accuracy of the construct and facilitate the incorporation of the desired fragment
into the plasmid (Hanahan, 1983).
Based on the information provided by the restriction analyzer, the BamHI enzyme cuts at two
different sites on genomic DNA (180 and 2865), producing large fragments that appeared as a
smear in the gel electrophoresis images (Sorek et al., 2013). HindIII was introduced to cut at an
additional site (4130) on genomic DNA, increasing the likelihood that the fragment containing
the rspL gene would be successfully incorporated into the plasmid (Chen & Dubnau, 2004).
In summary, the construction of a genomic library using E.coli Top10 genomic DNA and pUC19
plasmid DNA encountered several challenges throughout the experiment, including low DNA
concentrations, contamination, and inefficient transformation (Inoue et al., 1990). The
introduction of the HindIII enzyme in addition to BamHI for restriction digestion was expected
to increase the probability of obtaining a fragment containing the rspL gene (Cirino et al., 2003).
Furthermore, troubleshooting in each area of competent cells preparation, ligation reaction,
and transformation protocol can assist in identifying and rectifying these challenges, such as reevaluating the insert-to-vector ratio, incubation time, and temperature for the ligation reaction
or testing and optimizing various transformation conditions (Hanahan, 1983).
For this experiment, the most likely problem would be with transformation. It could be
optimized and modified by making changes mentioned below:
a. Temperature and duration: Confirm that the heat shock conditions (e.g. 42°C for 30-45
seconds) are appropriate for your cells (Inoue et al., 1990).
b. Recovery duration: Allow adequate time (usually 1 hour) for the cells to recover and express
the antibiotic resistance marker (Sorek et al., 2013).
c. Optimal antibiotic concentration: Ensure the appropriate concentration of streptomycin for
selection is used on the plates. Too high or low of a concentration may result in inadequate
selection (Chen & Dubnau, 2004).
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In addition to the technical challenges faced during the genomic library construction, human
errors could also have contributed to the unsuccessful or inconclusive results. These errors can
occur in sample handling, reagent preparation, equipment usage, and protocol execution. Such
errors may include:
1. Cross-contamination: During handling of samples, there is potential for cross-contamination
between different DNA samples or between the samples and reagents, leading to undesired
results (Inoue et al., 1990).
2. Reagent preparation: Errors can occur while preparing various reagents such as buffers,
enzyme solutions, or antibiotics. These could include inaccurate measurement or mixing of
components, resulting in incorrect concentrations that can affect experimental outcomes
(Sorek et al., 2013).
3. Pipetting errors: Inaccurate pipetting can lead to incorrect volumes being dispensed, resulting
in the incorrect sample or reagent concentration, which affects the outcome of experiments
(Hanahan, 1983).
4. Equipment usage: Improper operation, maintenance, or calibration of laboratory equipment
can generate unreliable or inconsistent results. For instance, inaccurate temperature settings
for incubation or heat shock can affect the transformation efficiency (Chen & Dubnau, 2004).
To minimize human errors, it is essential to maintain good laboratory practices, maintaining
proper organization of the workspace, labeling samples accurately, and double-checking
reagent concentrations. Additionally, regularly calibrating and maintaining equipment,
following established protocols carefully, and incorporating appropriate controls into
experiments will help reduce the occurrence of human errors and enhance the overall success
of the genomic library construction process.
Future experiments can investigate alternative restriction enzymes, plasmid vectors, and
competent cell strains to improve the overall success of the genomic library construction
process (Cirino et al., 2003). Moreover, ensuring a suitable recovery time for transformed cells,
providing a nutrient-rich medium during recovery, using appropriate plating methods and
conditions, and including positive and negative controls in each transformation experiment will
aid in enhancing the efficiency of the genomic library construction and subsequent functional
selection of the streptomycin resistance-conferring rspL gene (Hanahan, 1983).