2. Introduction
Laccases are multicopper oxidases that catalyze the oxidation of a number of substrates
with the reduction of molecular oxygen to water 1. These enzymes are a part of a broad group
polyphenol oxidases that contain copper atoms in the catalytic center2. Laccases are oxidases that
oxidize polyphenols, methoxy-substituted phenols, aromatic diamines, and a wide range of other
compounds3. Laccases are found to be produced by a variety of species including, plants, fungi,
insects and bacteria. Particularly laccases are plentiful in a variety of white rot fungi involved in
lignin metabolism. Lignin is a polymer of monolignins that is derived from wood and secondary
cell walls of plants4. Fungal laccases have been found to carry out a wide range of roles
including pathogen/host interaction, stress defense, and lignin degradation. Fungal laccases have
a higher redox potential as compared with plant or bacterial laccases and are therefore useful in
numerous biotechnological applications.
Laccases have gained substantial attention for their involvement in transforming phenolic
compounds including polymeric lignin and humic substances. These laccases are able to reduce
oxygen to water by a phenolic substrate. The low substrate specificity that laccases display
among other properties make it useful in biotechnology applications. Unlike peroxidases,
laccases do not need addition or synthesis of a low molecular weight cofactor like hydrogen
peroxide as its cosubstrate. This is due to the fact that there is plenty of oxygen in the
1 Giardina,Paola,et al."Laccases:A Never-Ending Story." Cellular & Molecular LifeSciences 67.3 (2010): 369-
385. Academic Search Complete. Web. 4 May 2015.
2 Brijwani,Khushal,Anne Rigdon, and Praveen V. Vadlani."Fungal Laccases :Production,Function,And Applications
In Food Processing."Enzyme Research (2010):1-10. Academic Search Complete. Web. 4 May 2015.
3 P. Baldrian,“Fungal laccases-occurrenceand properties,”
FEMS Microbiology Reviews,vol. 30, no. 2, pp. 215–242,2006
4 Lebo, Stuart E. Jr.; Gargulak,Jerry D. and McNally,Timothy J. (2001). "Lignin". Kirk‑Othmer Encyclopedia of
Chemical Technology. John Wiley & Sons, Inc.doi:10.1002/0471238961.12090714120914.a01.pub2.Retrieved
2007-10-14.
3. surrounding that can be used as its substrate 5. The copper in laccases can be bound to several
sites depending on which fungus produces it. There are Type 1, 2 and 3 binding sites that place
the copper centered bound to imidazole side chains.
Figure 1. Crystal Structure of fungal laccase with copper tri-nuclear arrangement in center6.
Laccases offer broad substrate specificity and ability to oxidize an extensive amount of
xenobiotic compounds including many phenolics. This ability of certain laccases make it a very
promising candidate for application in bioremediation. In a world that is constantly being
polluted by synthetic substances, the search for enzymes that are able to restore the natural
environment is a topic of great interest. Laccases are also involved in the development of
biofuels and have been researched as the cathode in enzymatic biofuels. Laccases have been
labeled as industrial catalysts with applications is textile dyeing, wine cork making, and many
other environmental, diagnostic and synthetic uses7.
The research conducted in the University of Houston-Downtown over the Spring 2015
semester, involved the isolation and amplification of RNA from laccase producing fungi. This
5 Baldrian,Petr."Fungal Laccases-occurrenceand Properties."Federation of European Microbiological Societies 30
(2005): 215-42.Print.
6 "The Armstrong Research Group." The Armstrong Research Group. Web. 4 May 2015.
<http://armstrong.chem.ox.ac.uk/laccase.html>.
7 Xu, Feng (Spring 2005)."Applications of oxidoreductases:Recent progress". Industrial Biotechnology (Mary Ann
Liebert, Inc.) 1 (1): 38–50. doi:10.1089/ind.2005.1.38.ISSN1931-8421.
4. study focuses on the physiological ecology of Coastal Prairie fungi, and the functional diversity
and characteristics of laccase enzymes produced by approximately 150 ascomycetes isolated
from prairie soils. The broad substrate specificities of laccases is the basis for their current and
potential uses as industrial catalysts. Screening of more than 100 of the Coastal Prairie isolates
for the presence of laccase DNA sequences and enzyme uncovered evidence of laccases in many
of the soil isolates. The research conducted involved using a technique known as Rapid
Amplification of cDNA Ends (RACE) PCR, to amplify laccase RNAs from a subset of laccase-
producing fungi which were converted to cDNA . The project would have also involved the
cloning for expression and characterization of laccases.
The plan was as follows and involved a number of steps. The first step involved isolation
of total RNA after induction of lacasse with veratryl alcohol. The next step involved
dephosphorylating non-mRNA and decaping mRNA to leave mRNA with only a 5’ P cap. This
was to be followed by the addition of RNA oligo to 5’ end of mRNA. This would make the first
strand of cDNA via 3’ oligo. This would involve the use of a single stranded cDNA which had a
5’ and 3’ oligo attached, each having unique primer sequences. This would then be treated with
RNas to remove RNA thus leaving the 1st strand cDNA. The 5’ and 3’ ends would then be
amplified of laccase cDNA only. Both ends would then be sequenced to design laccase primers.
Materials and Methods
All the materials utilized in this research experiment were provided by the University Of
Houston Downtown Department of Natural Sciences. RNA isolation kit was provided by
QIAGEN as the RNeasy Mini kit. Materials included inside kit were RNeasy Mini spin columns,
collections tubes, 1.5 ml and 2 ml, Buffer RLT, Buffer RW1, Buffer RPE, and RNase-free water.
5. Materials for Gene Racer kit were provided by Invitrogen and included, CIP, 10X CIP buffer,
10X TAP buffer, SuperscriptIII RT, T4 RNA ligase, 10mM ATP, Phenol/Chloroform, Mussel
Glycogen, 3M Sodium Acetate, Gene Racer 5’,3’ primers, nested primers, control Hela Total
RNA, Oligo dT primer, dNTP mix, 5x RT buffer, GeneRacer Oligo dT Primer, 100 mM dNTP’s.
Selection and Growth of Fungi
Nine different known laccase producing fungi were selected for RNA isolation. The
different fungi contained different identification numbers. The fungi were renumbered to make
labeling easier to accomplish.
Table 1. Fungal Sample Identification
Fungi Identification Number Spring 2015 Number Designation
9F3-44-1 1
9F3-45-1 2
9F6-35-1 3
9U14 4
9U12 5
9F2-31-1 6
9U4 7
9U16 8
9F9-62-1 9
Once fungi were selected, one square was cut and placed into a fresh plate of PDA. Three
quare samples were placed into 3 different plates for each fungi sample 1-9. The fungi was
6. allowed to grow at room temperature for the remaining of the semester. The preparation of the
fungi used for RNA extraction involved the use of Malt extract agar. For this procedure Bacto
Malt Extract, Peptone and 99.5 % glucose was prepare and 25ml of this was placed into
Erlenmeyer flasks. These flasks were placed on a autoclave machine to remove any bacteria.
Duplicate samples were made by placing 4-5 square samples of fungi into each of the
Erlenmeyer flasks. They were placed on a shaker at 200 rpm for 6 days. On day 6 veratryl
alcohol was placed on Erlenmeyer Flasks, after which it was allows to shake for an additional 24
hours before RNA extraction proceeded.
Fungal Sample Grind
Fungi samples from Erlenmeyer Flasks were placed into large centrifuge tube and spun
down at 40, max speed for 20 minutes. This allowed fungi sample to stick to bottom of centrifuge
tube to make it easier for RNA extraction. Supernatant was filtered away as samples stuck to
filter paper. Bench was sprayed with RNAse Away liquid along with mortar and pestle to make
sure any ribonucleases were eliminated. RNAeasy procedure was inspected to make sure all
reagents were present and plentiful. Various tubes and columns were paced on tube holders and
numbered with appropriate designations. Gloves and spatula were sprayed multiple times
through the procedure. Liquid nitrogen from lab stockroom was placed on liquid nitrogen
holding container and taken to lab bench. Mortar was placed close to edge of table as liquid
nitrogen was poured into mortar. Liquid nitrogen was allowed to boil for a few moments before
fungi sample was dropped inside. Pestle was used to carefully grind sample as it froze. As
nitrogen began to boil over the sample was hit with the pestle aggressively, grinding the sample
to fine powder residues. Small weight boats were used to transfer powder sample to scale. Less
than 100 mg was used and placed into 1.5ml RNase free tubes containing Buffer RLC. The
7. sample was vortexed and placed into ice. The same procedure to grind fungi sample into fine
powder was done for each sample.
RNA extraction
The lysate from all samples were transferred into QIAshredder spin column and
centrifuged for 2 minutes at full speed. The supernantant was transferred into a new 1.5 ml
RNase free tube were 0.5 volume of 96-100% ethanol was added and mixed. This was
transferred to an RNeasy spin column with a 2 ml collection tube were it was spun for 15
seconds at 10,000 rpm. Supernatant was discarded and 700μl of buffer RW1 was added. This
again was spun for 15 seconds at 10,000 rpm where flow-through was discarded. There was an
addition of 500μl of buffer RPE to the RNeasy spin column where it was spun for 15 seconds at
10,000 rpm. The column was placed in a new 1.5 ml collection tube where 50μl of RNase-free
water was added directly to the column membrane. This was centrifuged for 1 minute at 10,000
rpm to elute the RNA. The tubes were placed in -800 C fridge were subsequent steps would
follow.
RNA concentration determination and Gel Electrophoresis.
Before proceeding to next steps it was necessary to determine RNA concentration
obtained. There had to be sufficient RNA concentration to proceed. This was done by taking
frozen samples and thawing them out on ice. The spectrophotometer used a single cubet with a
2mm cap. Cubet was cleaned with 70% ethanol and allowed to dry. RNase free water was used
as a blank. Once blank and appropriate software functions were determined, 1 μl samples were
pipetted on cubet and cap placed. After each sample was pipetted on cubet it was closed and
“Go” button was hit. After each sample was done, the cubet was cleaned with ethanol to remove
8. any residues. Data was given in tables of Absorbance at 260.0nm, 280.0 nm, Bkg at 320.0 nm,
260/280, 280/260 ratios, protein and nucleic acid concentrations in μl/ml. Gel electrophoresis
was conducted with a 1% gel. The gel consisted of 1.2 grams of agarose in 120ml of TAE 1X
buffer. The agarose was poured on an Erlenmeyer Flask along with the TAE buffer. This was
heated for approximately 45 seconds making sure agarose was completely dissolved. The gel
was allowed to solidify. The samples were loaded as 10ul total volume consisting of purified
water, dye, and RNA sample. The ladder was a 1kb Lambda DNA. The gel was allowed to run
at 120 Volts for approximately 1 hour. The gel was then taken to UV Transilluminator for
imaging of results.
Gene Racer
Dephosphorylatin RNA
The first step was to setup the dephosphorylation procedure which consisted of a total of
10ul volume reaction. The reaction included the following reagents mixed in a 1.5 ml tube. 10X
CIP buffer, RNase out, CIP, and RNase free water. This was mixed and incubated at 500 C for 1
hour, after which it was placed on ice. To precipitate RNA, 90 ul of Rnase free water and 100ul
of phenol:chloroform were added and vortexed for 30 seconds. This was centrifuged for 5
minutes at room temperature. Top aqueous phase was transferred to microcentrifuge tube after
which 2ul of mussel glycogen, 10ul of 3M sodium acetate, and 220ul of 95% ethanol was added
and vortexed. This was frozen on ice for 10 minutes. To pellet RNA it was centrifuged at max
speed for 20 minutes at 40C. The position of the pellet was noted and supernatant removed.
There was an addition of 500ul of 70% ethanol and centrifuged at max speed for 2 minutes at
40C. Again the position of pellet was observed and ethanol removed. This was centrifuged for 2
minutes at room temperature. The pellet was resuspended in 7ul RNA-free water.
9. Removing the mRNA Cap Structure
The removing of mRNA cap structure required the following reagents, 10X TAP buffer, RNase
out, and TAP. This was mixed to a total of 10ul, vortexed and centrifuged. This was incubated at
370C for 1 hour. After incubation, 90ul of RNase free water and 100 ul of phenol:chloroform was
vortexed vigorously for 30 seconds. This was centrifuged at max speed for 5 minutes at room
temperature. Top layer was transferred to new tube. 2ul of 10mg/ml mussel glycogen, 10 ul 3M
sodium acetate and 220 ul of 95% ethanol was added. Sample was frozen in -800C overnight.
The sample RNA was pelleted on microcentrifuge for 20 minutes at 40C. Supernatant was
carefully removed and 500ul of 70% ethanol added. This was centrifuged for 2 minutes at 40C.
Again supernatant was removed and pellet resuspended by adding 7 ul of RNase free water.
Ligating the RNA oligo to Decapped mRNA
Addition of 6ul of dephophorylated, decapped RNA was added to RNA oligo. This was
incubated at 650C for 5 minutes. This was placed on ice for 2 minutes and centrifuged briefly.
The following reagents were then added, 10X Ligase Buffer, 10mM ATP, RNase Out, t4 RNA
ligae and incubated at 370C for 1 hour. After incubation 90ul of Rnase free water and 100 ul of
phenol:choloform was added and vortexed for 30 seconds. This was centriguged for 5 minutes at
max speed. Top layer was transferred to new tube and 2 ul of mussel glycogen 10ul of sodium
acetate, and 220 ul of ethanol were added and vortexed. The next following protocol is the extact
same as performed for the precipitation of RNA mentioned above.
Reverse Transcribing mRNA
The cloned AMV RT reaction was as follows, 1ul of desired primer and 1 ul of dNTP was added
to ligated RNA. This was incubated at 650C for 5 minutes. This was chilled on ice for 2 minutes
10. and centrifuged briefly. The following reagents were mixed, 5x RT buffer, Cloned AMV RT,
sterile water, and Rnase out and incubated at 450C for 1 hour. This was then incubated at 850C
for 15 minutes and centrifuged. This was stored in -800C over night. The superscript III RT
reaction involved poly-T primers, dNTP mix, and distilled water. This was incubated at 650C for
5 minutes, followed by chilling on ice for 1 minute. 5X strand buffer, 0.1 M DTT, RNase out,
and Supercript II RT was added and mixed. This was incubated at 250C for 5 minutes. This was
incubated at 500C for 60 minutes followed by 15 minutes at 700C. This was chilled on ice for 2
minutes and centrifuged at max speed. One ul of RNase H was added to reaction mix and
incubated at 370C for 20 minutes. This was centrifuged and stored in -800C.
Amplification of cDNA Ends
Following gel electrophoresis and spectrophotometer analysis it was decided to proceed
with PCR reaction. This involved 1ul of 5’ primer, 1ul of 6ARI, 15 ul of DNA and 8 ul of water.
The reaction PCR was performed based on the parameters in the handout.
Results
The experiment was initiated with the RNA extraction of the first four samples. There
was however a mistake made with use of a QIAshredder instead of an RNeasy spin column. For
this reason the initial samples 1-4 did not obtain any results. The Gel electrophoresis conducted
on those samples did not reveal any bands. This is something that was to be expected being that
the RNA was not captured on the column and therefore not eluted out. The RNAspin columns
were located in different bag and it was assumed the QIAshredders were the spin columns.
Samples 5-8 were conducted with the appropriate RNA spin column and concentrations but were
conducted with dry ice instead of liquid Nitrogen. The University of Houston had run out of
11. liquid nitrogen on the day those particular isolations were conducted. It was decided to conduct
isolations using dry ice from the Chemistry Stockroom. The concentrations for them are as
follows.
Table 2. RNA Concentration Samples 5-8
Sample # Abs
260nm
Abs
280nm
BKG 320.onm 280/260 260/280 Protein
ug/ml
NucAcid
ug/ml
5 -0.0356 -0.0391 -0.0251 0.7503 1.3329 -28.0148 -21.0185
6 0.8499 0.8582 0.8931 1.2358 0.8092 -69.8789 -86.3583
7 0.0804 0.0497 0.0357 3.1850 0.3140 28.0727 89.4104
8 0.0665 0.0347 0.0242 4.0458 0.2472 20.8752 84.4562
The concentration numbers appear very low with some samples obtaining negative numbers. It
was decided to conduct a second concentration determination with samples 6-7.
Table 3. RNA concentration Samples 6-8
Sample # Abs
260nm
Abs
280nm
BKG 320.onm 280/260 260/280 Protein
ug/ml
NucAcid
ug/ml
6 0.0442 0.0441 0.0411 1.0403 0.9613 5.88 6.12
7 0.0847 0.0549 0.0249 1.9901 0.5025 60.09 119.60
8 0.0453 0.0335 0.0192 1.8293 0.5467 28.46 52.07
It was determined that not enough Nucleic acid concentrations were obtained and therefore more
RNA isolations were conducted with liquid nitrogen in order to obtain favorable results. Its important to
note that these concentration were low as compared to previous isolations conducted by other students in
past semesters.
12. A second round of RNA isolations were conducted on duplicate samples that had been prepared
using the same technique as mentioned above. This time 5 samples were conducted using the correct
columns as well as liquid nitrogen. The results are as follows:
Table 4. RNA Concentrations Samples 1-5
Sample # Abs
260nm
Abs
280nm
BKG 320.onm 280/260 260/280 Protein
ug/ml
NucAcid
ug/ml
3 -0.0436 -0.0540 -0.0677 1.7565 0.5693 34.3121 60.2705
4 0.0209 0.0617 -0.0240 1.7470 0.5724 64.2638 112.2712
5 0.0009 -0.0517 -0.0977 2.1436 0.4665 115.0172 246.5507
6 0.0215 -0.0072 -0.0411 1.3487 0.5404 84.7717 156.7205
7 0.0231 -0.0168 -0.0546 2.0564 0.4863 94.5110 194.3566
These concentration numbers were deemed suitable for the Gene Race procedure and it
was decided to proceed forward with samples number 5 and 7.
.
Figure 2. Gel electrophoresis of samples 5-7. Samples 5 and 7 show 2 bands consistent with
RNA 18S and 11S subunits.
13. Based on Gel electrophoresis results it was decided to obtain more concentration numbers on
samples 5-7.
Sample # Abs
260nm
Abs
280nm
BKG 320.onm 280/260 260/280 Protein
ug/ml
NucAcid
ug/ml
5 -0.0050 -0.0104 -0.0173 -0.0173 1.7855 17.2552 30.80
6 1.2344 1.2365 1.2365 1.2346 -0.0863 4.7982 -0.4140
7 0.7528 0.7301 0.7301 0.6749 1.4104 138.0295 194.6720
These 2 samples in combination with 3 other samples obtained from Jonathan Cheatham,
the Gene racer procedure was initiated. A total of 5 samples including samples 2, 3,4,5 and 7
were the selected ones that obtained sufficient RNA concentrations to continue.
The GeneRacer procedure should have been undertaken on a few days but was extended
for a number of reasons due to shortage of time and Spring Break. The initial procedure called
for the use of 10X CIP buffer which was missing from the kit. The 10X CIP buffer was therefore
made with 0.5 M Tris HCL and 1mM EDTA. The pH was adjusted to 8.5 and used in the
subsequent steps. RNase Out was replaced with RNase inhibitor and DEPC water was replaced
with RNase free water. The procedure was followed as mentioned in lab manual with a series of
stop points along the way. It was made sure the RNA was not stored in DEPC water in the freeze
points. In total the procedure was halted 5 times in the store overnight points. The final step
before PCR was initiated was the reverse transcribing mRNA protocol. It was at this point that
concentrations were checked under the spectrophotometer. The concentrations were very low
and dismal.
14. It was at this point that it was decided to proceed with the amplification of cDNA ends
anyways and see if it was possible to obtain results. The PCR procedure involved the use of PCR
beads, DNA from samples, primers, and water. The PCR was conducted on a thermo cycler and
allowed to remain until the following day when they were removed and placed on fridge. One μl
of sample from each fungi was taken to spectrophotometer and observed for results.
Table 5. PCR results
Sample # Abs
260nm
Abs
280nm
BKG 320.onm 280/260 260/280 Protein
ug/ml
NucAcid
ug/ml
2 0.2654 0.1421 -0.0050 1.8382 0.5440 294.2712 540.9243
3 0.7357 0.4359 0.0215 1.7235 0.5802 828.8643 1428.5446
4 1.0138 0.6340 0.0686 1.6719 0.5981 1130.6991 1890.3698
5 0.7856 0.4560 -0.0137 1.7016 0.5877 939.5483 1598.7758
7 0.8170 0.5183 0.0392 1.6233 0.6160 958.2827 1555.5545
These numbers appear very promising with high Nucleic Acid concentrations. AT this
point a gel electrophoresis was conducted to visualize the bands that should be there. The gel
was created as 0.8% and run at 140 Volts for approximately 1 hour.
Figure 3. 0.8% gel with PCR reactions. Samples 2,3,4,5,7.
15. The results of gel show two lower bands that correspond to the primers utilized. There
appears to be no other bands in the gel. These results indicate that the Gene Racer procedure was
a failure. There are no observable bands above the seen primers. At this point it was decided to
check the RNA concentration on the original samples and proceed again if possible with the
Gene Racer protocol. The samples were checked for concentrations and revealed extremely low
numbers in the 8ug/ml range.
With time running out, it was determined that regroqing the original fungi would take too
much time. AT this point it was decided to take samples from Jonathan Cheetham and perform
extractions of those. Jonathan had a total of 8 samples on centrifuge columns from which
extractions would take place. Four samples were then taken and RNA isolation as mentioned
above was done. RNA concentrations were taken to spectrophotometer for viewing. Results are
as follows.
Table 6. RNA extraction concentration. Jonathan samples
Abs
260nm
Abs
280nm
BKG 320.onm 280/260 260/280 Protein
ug/ml
NucAcid
ug/ml
0.0045 0.0023 -0.0003 1.8733 0.5338 5.0609 9.4806
0.0058 0.0030 -0.0001 1.8728 0.5340 6.2931 11.7855
0.0065 0.0036 0.0002 1.8772 0.5327 6.6899 12.5586
0.0070 0.0039 0.0003 1.8438 0.5424 7.1812 13.2407
The concentrations are very low and clearly could not be used in the Gene Racer
experiment. The following 2 extractions were conducted with a slight modification. The crushed
sample was added to the 1.5 ml tube and dipped for a few seconds in liquid nitrogen and taken to
16. -80 freezer immediately and placed on ice. This was done for 2 samples after which
concentrations were checked. The results revealed similar results from the ones seen above.
Discussion
Overall the goal of this research project was not met and could be due to a number of
reasons. One can definitely point to the lack of experience in lab techniques and RNA isolation
as the main cause for failure. This was first observed in the initial process with the use of the
QIAshredder instead of the RNeasy column. It is however understood that isolation of RNA is a
very delicate process that can be difficult to accomplish even with experience technicians. The
first run at RNA isolation also hit a problem with the use of dry ice instead of liquid nitrogen.
This was an experimental procedure that had to be done due to lack of available liquid nitrogen.
This however does not mean that successful RNA isolation was not possible. There is no
question that freezing and crushing the fungi with dry ice required more patience and more
crushing to get powdered residues. The results from this procedure showed no bands on the gel
and low concentration on the spectrophotometer. However this procedure served as a practice
run that would give confidence and experience when eventually the liquid nitrogen extractions
were conducted. It was not such a bad thing to perform the isolations several times. The second
run at isolations with the correct columns and use of nitrogen revealed desirable results. The gel
showed 2 bands and concentrations were not great but sufficient enough to continue.
The Gene racer protocol initiated with the absence of CIP buffer. This was created frorm
EDTA and Tris-HCL. Is it possible that this buffer created was not adequate enough to work?
This is definitely a possibility that should be considered. The experimental flowchart reveals this
complex procedure to be accomplished in 2 days. This however was something that was
ambitious and extremely challenging given the amount time in hand. Overall the whole
17. procedure was halted a total of 5 times in the freeze overnight points along the way. The stop
points reveal the caption, “You may proceed to the next step or store at -20 degrees C overnight.
Note: Do not store the RNA in DEPC water. Store RNA in ethanol at -200C”. Is it possible that
there was still water in the tube? This can certainly be a possibility. The procedure called for the
addition of water and phenol:chloroform followed by the transfer of the top layer to a new tube.
There is no question that ethanol was present but it is also possible that small amounts of water
remained. Another aspect that was hard to accomplish was the removal of supernatant after
centrifugation in the precipitating RNA protocol. All the supernatant was remove leaving an
empty tube. This is the portion where it was possible to accidentally throw away the RNA
present. There was after all no way to determine if there was RNA present in the tube. There
was also the issue of the incubation periods in the reverse transcribing protocol. Some of the
procedures required the use of 3 incubation with different temperatures. The machine in the
laboratory only had 2 slots to incubate at 2 different temperatures. Sometimes the adjusting to a
different temperature required the sample to stay longer in one particular temperature for longer
than required to wait for the right temperature to set.
There was also the mistake of not checking to see the concentration numbers after each
step in the protocol. There should have been repeated checks for concentrations at each stopping
point or at the conclusion of each segment. This was a mistake that could have been easily
avoided. The reasoning behind this decision was to limit the amounts of thaws of RNA samples.
However checking the amount of concentration before going forward was a necessary step that
was not accomplished.
18. Conclusions
Overall the experiment was not a success due to a number of possibilities. Lack of
experience and poor lab techniques played a major role in the outcome. The rush to obtain results
and put all eggs on one basket ultimately ended in failure. There is no question that better
communication between the working partners and leading investigator should have taken place.
It is however understood that this is a very delicate and difficult project that will require the
upmost expertise and brain knowledge to successfully accomplish.
19. References
Giardina, Paola, et al. "Laccases: A Never-Ending Story." Cellular & Molecular Life Sciences
67.3 (2010): 369-385. Academic Search Complete. Web. 4 May 2015.
Brijwani, Khushal, Anne Rigdon, and Praveen V. Vadlani. "Fungal Laccases: Production,
Function, And Applications In Food Processing." Enzyme Research (2010): 1-10. Academic
Search Complete. Web. 4 May 2015.
P. Baldrian, “Fungal laccases-occurrence and properties,”
FEMS Microbiology Reviews, vol. 30, no. 2, pp. 215–242, 2006
Lebo, Stuart E. Jr.; Gargulak, Jerry D. and McNally, Timothy J. (2001). "Lignin". Kirk‑Othmer
Encyclopedia of Chemical Technology. John Wiley & Sons, Inc.
doi:10.1002/0471238961.12090714120914.a01.pub2. Retrieved 2007-10-14.
Baldrian, Petr. "Fungal Laccases-occurrence and Properties." Federation of European
Microbiological Societies 30 (2005): 215-42. Print.
"The Armstrong Research Group." The Armstrong Research Group. Web. 4 May 2015.
<http://armstrong.chem.ox.ac.uk/laccase.html>.
Xu, Feng (Spring 2005). "Applications of oxidoreductases: Recent progress". Industrial
Biotechnology (Mary Ann Liebert, Inc.) 1 (1): 38–50. doi:10.1089/ind.2005.1.38. ISSN 1931-
8421.
