Slide 1: Title Slide- Troubleshooting and Next Steps in Genomic Library ConstructionSlide 2: Overview- Summary of project and challenges encountered- Importance of troubleshooting and optimizationSlide 3: Low DNA Concentrations- Effect on gel electrophoresis results- Solutions: Clean up methods and optimizing digestion reactionsSlide 4: Contamination Issues- Effects on transformation results- Solutions: Proper lab practices and appropriate controlsSlide 5: Inefficient Transformation- Errors in competent cell preparation, ligation reaction, and transformation protocol- Solutions for optimization in these areas discussed in the following slidesSlide 6: Ligation Reaction Optimization- Re-evaluating insert-to-vector ratio- Adjusting incubation times and temperatureSlide 7: Transformation Conditions Optimization- Testing different temperature and duration settings for heat shock- Ensuring optimal recovery time for transformed cells- Using appropriate antibiotic concentrations for selectionSlide 8: Alternative Experimental Approaches- Investigating alternative restriction enzymes, plasmid vectors, and competent cell strainsSlide 9: Ensuring Proper Controls and Recovery Conditions- Importance of including positive and negative controls in every experiment- Providing nutrient-rich media during recovery and proper plating methodsSlide 10: Conclusion- Troubleshooting and optimization are critical for successful genomic library construction- Future experiments can enhance efficiency by considering alternative enzymes, plasmids, competent cells, and improving techniquesSlide 11: Next Steps1. Re-evaluate and optimize the transformation process2. Test alternative restriction enzymes, plasmid vectors, and competent cell strains3. Ensure appropriate controls, recovery conditions, and nutrient-rich media are used in each experiment4. Continue troubleshooting and optimizing other areas of the experiment as necessarySlide 12: Acknowledgements and References- Thank any individuals involved in the project- List relevant references used throughout the experiment and presentation

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Background
This portion of the lab section involved creating a genomic library using a streptomycin
resistant strain of E.coli top10 (4537 bp) as the genomic DNA which will be incorporated into
puc19 plasmid (2686 bp) vector as proposed earlier in PART B proposal document.
To achieve the proposed plan, E.coli strain top10 (4537 bp) will serve as the genomic strain that
will be the source of streptomycin resistance rspL gene. The FastDigest HindIII (thermo
scientific) digested fragment will be insert into a digested pUC19 plasmid vector (2686 bp)
utilizing ligation process. Once ligated, the product will be transformed with Mach1 chemically
competent cells (O.D. value = 0.77). To confirm the success of ligation the transformed product
will be plated on Ampillicin/X-gal/IPTG plates and the white colonies will then be streaked onto
streptomycin plates to verify the rspl gene streptomycin resistance property.
As per the proposal, digesting the bacterial strains with similar digestion enzyme (HindIII) prior
to ligation followed by transformation would have ideally given the genomic library however,
throughout the project certain decisions were changed based on the results which will be
thoroughly covered in this document along with the solutions taken to troubleshoot the
mistakes.
Methods/Materials
Isolation of genomic DNA and plasmid DNA:
To start with the procedure of creating a genomic library from E.coli strain Top10 and plasmid
pUC19, 5 mL genomic DNA (Top10) and 3 mL plasmid pUC19 was isolated using GeneJET
genomic DNA purification kit (lot 01319594) and GeneJET plasmid Mini prep kit (lot 01218820)
respectively.
Table 1: Observed concentrations of isolated genomic material using SimpliNano.
Genomic material A260/A280 A260/A230 DNA Conc. (ng/µL)
Top10 1.988 1.644 33.2
pUC19 1.866 1.211 17.2
The above table summarizes the observed concentration data for isolated genomic material on
Feb 21, 2023. The volume of sample loaded in SimpliNano was 2 µL for both DNA. Elution
buffer(01177350 and 00991597) from both respective isolation kits were used as a blank on
SimpliNano.
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Restriction Enzyme Digestion:
As per part A proposal our group wanted to use HindIII enzyme of restriction enzyme digestion
but BamHI was used instead. This was changed because the aim was to check whether BamHI
will show same digestion or similar results with strainTop10 of E.coli as it was the case with K12
strain.
Both genomic DNA from Top10 and plasmid DNA of pUC19 were digested with thermos
scientific FastDigest BamHI enzyme in second week of this project (March 8, 2023) and the
reagent volumes used are listed in the table given below.
Table 2: Reagents for restriction enzyme digestion of genomic material.
Digestion Reagents pUC19 Top10 (rspL gene)
Genetic material 2 µL 10 µL
Fast digest buffer 2 µL 2 µL
BamHI (Fast Digest Enzyme) 1 µL 1 µL
PCR Grade Water 30 µL 22 µL
Total Reaction volume 35 µL 35 µL
This table summarizes the reagent recipe used for first restriction enzyme digestion of genetic
materials used to create the genomic library. The above listed both digestion reactions were
incubated in 37 °C water bath for 30 minutes before heat inactivation at 80 °C for 10 minutes.
Gel electrophoresis to verify digestion from band sizes:
This gel electrophoresis was performed on same day in order to confirm whether the digestion
was successful or not and whether the isolated genomic DNA contains the rspL gene. To
prepare this, 1% gel was prepared using 500 mg BIOTECHNOLOGY GRADE Agarose ITM powder
(Lot : 19H2056197) in 50 mL 1X TAE buffer with 2.5 µL INtRON RedSafeTM (0008-090501.52) for
visualization.
1Kb DNA ladder RTU (Froggo-BIO, BIO-HELIX LOT 14007210104) was used as a standard with
Thermo Scientific 6X loading dye (LOT 00799325). 10 µL of digested genomic was loaded into
the gel. Gel was run for 20 minutes at 120V. When analyzed under UV light(Midi Blue LED
Transilluminator), there were no bands visible.
DNA clean-up:
Since gel electrophoresis results indicated no bands it was decided with the help of lab
instructor to perform a DNA clean up for genomic to concentrate it more.
Originally isolated genomic Top10 DNA was cleaned up using “Thermo Scientific GeneJET Gel
Extraction and DNA clean up kit” the following week ( March 14, 2023). A small change was
introduced in original clean up protocol which stated “10 µL of Elution buffer” to elute which
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was updated to “15 µL of Elution Buffer”. Concentration of genomic DNA was checked on
SimpliNano after clean up which indicated the DNA was lost in clean up process.
Table 3: Concentration of genomic DNA (Top10) analyzed on SimpliNano after DNA clean up.
Cleaned up genomic Top10 DNA
A260/A230 0.826
A260/A280 1.609
ng/ µL 12
The above table lists the concentration of originally isolated genomic DNA of strain Top10 after
clean-up using Thermo Scientific GeneJET Gel Extraction and DNA clean up kit (LOT 2599306).
Volume of sample loaded on SimpliNano was 2 µL and the blank used was the elution
buffer(LOT 01307087) from the DNA clean up kit.
New isolated genomic Top10 DNA provided by Vanessa:
Since the whole genomic DNA was lost therefore another vial of isolated genomic was provided
by lab instructor. The concentration of new genomic DNA was analyzed on SimpliNano which
was then cleaned-up using the same clean up kit with same modification of 15 µL elution
buffer. After clean up the concentration was checked on SimpliNano and is stated in table
below.
Table 4: Concentration of new genomic Top10 DNA before and after clean-up.
Before clean up After clean up
A260/A280 1.598 1.794
A260/A230 1.143 2.188
ng/ µL 28.1 109.6
The above table summarizes the observed concentrations of new isolated genomic top10 DNA
provided by Vanessa. These concentrations refer to the DNA before using GeneJET genomic
DNA purification kit with its corresponding elution buffer as blank. Also after clean up using
Thermo Scientific GeneJET Gel Extraction and DNA clean up kit (LOT 2599306). Volume of
sample loaded on SimpliNano was 2 µL and the blank used was the elution buffer(LOT
01307087) from the DNA clean up kit.
New Digestion reaction set up with cleaned up genomic DNA:
The new digestion was performed using the cleaned up genomic DNA with higher
concentration. The table summarizes the reaction reagents and volumes.
Table 5: The reaction reagents for restriction enzyme digestion of both genomic material.
pUC19 Top10 genomic DNA
Concentration 17.2 ng/ µL 109.6 ng/ µL
Genomic material 4 µL 5 µL
Fast Digest Buffer 2 µL 2 µL
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Fast Digest Enzyme (BamHI) 1 µL 1 µL
PCR Grade water 28 µL 27 µL
Total reaction volume 35 µL 35 µL
The table gives the reaction volumes that were used to perform enzyme digestion of both
genetic materials used in this project to construct a genomic library. Both reactions were
incubated for half an hour in 37 °C water bath prior to heat inactivation at 80 °C for 10 minutes.
The digestion reactions were then stored in ice bucket until gel electrophoresis apparatus was
ready.
Another 1% gel electrophoresis:
Similar recipe of 1% gel electrophoresis was prepared using same supplies however since the
concentration of newly cleaned genomic DNA was significantly high, only 5 µL of cut and uncut
genomic along with cut plasmid with 6X loading dye was loaded.
The 1% gel was shared with another group with ladder in the center followed by uncut
genomic, cut genomic and cut plasmid from our samples. First half of the gel was utilized by
Mahan’s group. The gel was run for about 20-25 minutes at 120 V and the results were first
visualized under UV light (Midi Blue LED Transilluminator) and then under BIOrad software
which will be added in result section.
Ligation with only one reaction:
This was decided to check if the ligation was successful or not which was performed the
following week (March 21, 2023). To do so, a 1:1 ratio of cut plasmid pUC19 and cut genomic
top10 (insert) was ligated using T4 ligase (vector) accompanied with the use of PEG4000.
The following table summarizes the reaction reagents and volumes.
Table 6: Ligation reaction set up for digested insert and vector using T4 ligase.
Reaction reagents Ligation ratio (1:1)
Insert (digested genomic top10 DNA) 3 µL
Vector (digested pUC19) 3 µL
T4 Ligase Buffer (5X) 5 µL
T4 Ligase 2 µL
PEG4000 2 µL
PCR Grade water 10 µL
Total reaction volume 25 µL
This ligation reagent recipe was used to ligate the insert and vector achieved from restriction
enzyme digestion of genomic top10 and pUC19 with BamHI. This ligation reaction was
incubated overnight at 4 °C and heat inactivated after 24 hours at 65 °C for 10 minutes. The
ligated product was then stored at -20 °C until transformation.
Transformation with Mach 01 strain competent cells:
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Transformation of the ligated product was performed the same week (March 24, 2023). To
proceed with transformation, 4 vials of competent cells (50 µL each) were provided by Vanessa
along with 4 agar plates.
Goal was to plate one positive control, one negative control for validation of results with one
experimental in duplicates. Protocol of transformation was used to first thaw the competent
cells on ice (about 20 minutes) followed by setting up controls and experimental.
Table 7: Controls and experimental reaction set for transformation of ligated product.
Reaction Positive control Negative control Experimental
Reagents 50 µL competent
cells + 10 µL pUC19
50 µL of competent
cells
50
The transformation reactions were then incubated on ice for 30 minutes prior to heat shock at
42 °C for 30 seconds. The reactions were then placed on shaker for 1 hour until recovered.
Meanwhile, when 30 minutes were left while cells were recovering, 4 agar plates were spread
with 100 µL of 25 mg/mL Ampicillin, 40 µL of 20 mg/mL X-gal and 8 µL of 500 mM IPTG per
plate respectively. After successive completion of 1 hour recovery of cells, one little change was
introduced in which the cells were pelleted at 5000 rpm for 5 minutes. Instead of plating 200 µL
onto plates, 150 µL was plated for each to increase the concentration.
Once solidified, all 4 plates were incubated at 35 ° C for 24 hours. Plates were retrieved from
incubator the next day (March 25, 2023).
The results of transformation depicted signs of contamination and no white colonies were
present. On the assumption that contamination might have masked the ligation results.
New transformation using same ligated product:
The following week (March 28, 2023), the same ligated product was used for transformation
using same supplies as in previous section. No ligation control was set up as there was not
enough ligated product left. Following exact same procedure the transformation was
completed and plates were incubated at 35 ͦ C for 24 hours. Plates were retrieved from
incubator the next day (March 29, 2023).
The results suggested no success since no white colony was visible.
Digesting with BamHI and HindIII :
In the final week (April 4, 2023) of this project, a new digestion reaction was set up using two
restriction enzymes that is FastDigest HindIII (thermo scientific) and FastDigest BamHI (thermo
scientific).
The reaction reagents and volumes used are listed in the table given below
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Table 9: The reaction reagents for restriction enzyme digestion of both genomic material using
two enzymes.
pUC19 Top10 genomic DNA
Concentration 17.2 ng/ µL 109.6 ng/ µL
Genomic material 2 µL 2 µL
Fast Digest Buffer 1 µL 1 µL
Fast Digest Enzyme (BamHI) 2 µL 2 µL
Fast Digest Enzyme (HindIII) 2 µL 2 µL
PCR Grade water 3 µL 3 µL
Total reaction volume 10 µL 10 µL
The table enlists the reaction volumes that were used to perform enzyme digestion of both
genetic materials with thermos scientific FastDigest BamHI and HindIII. Both digestion reactions
were incubated for half an hour in 37 °C water bath prior to heat inactivation at 80 °C for 10
minutes. The digestion reactions were then stored in ice bucket until gel electrophoresis
apparatus was ready.
Gel electrophoresis to verify digestion of genomic material using 2 restriction enzymes:
While performing the digestion reactions one team member was setting up the 1% agarose gel
preparation. To do so, 500 mg BIOTECHNOLOGY GRADE Agarose ITM powder (Lot : 19H2056197)
was used in 50 mL 1X TAE buffer. 2.5 µL INtRON RedSafeTM (0008-090501.52) was added to the
mixture once cool enough.
1Kb DNA ladder RTU (Froggo-BIO, BIO-HELIX LOT 14007210104) was used as standard.
About 10 µL of the sample with 2 µL loading dye was used to load digested genomic top10 ,
digested genomic , undigested pUC19 plasmid , digested plasmid were loaded along with the
standard using Thermo Scientific 6X loading dye (LOT 00799325). Once the set up was ready
and the 30 minutes incubation was complete, the 1% gel electrophoresis was performed was 20
minutes at 120V. On completion the gel was visualized under UV light (Midi Blue LED
Transilluminator).
The gel results were not up to mark but since there was no time to carry out any other
procedures this project came to an end.
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 © 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 © ,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).
Conclusion
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In conclusion, the construction of a genomic library using E.coli Top10 genomic DNA and pUC19
plasmid DNA proved to be challenging due to several factors, including low DNA
concentrations, contamination, and inefficient transformation. Optimization of the
transformation process, selection of alternative restriction enzymes, plasmid vectors, and
competent cell strains could enhance the overall success of the genomic library construction
and subsequent functional selection of the streptomycin resistance-conferring rspL gene.
Future experiments should also emphasize proper controls, recovery conditions, and nutrientrich media to improve the efficiency of this process.