General Introduction To Molecular Biology LAB REPORT 26 August 2023 By Gulinky Lu Supervised By Dr Haiqing Yi, PhD Duke University Medical Centre Department of Pediatrics Abstract lol, imagine writing an abstract Contents 1 Culturing And Purification of Plasmid From Bacterial Cells 1 1.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Measured DNA Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 Restriction Digestion of pGFP And pGAA 1 2.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3 pGFP And pGAA Transfection of COS-7 Cells 3 3.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4 pGFP Expression 4 4.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 Assessing GAA Expression Through α -Glucosidase Activity And BCA Assays 5 5.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6 Genotyping of Mouse Tails 7 6.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 7 Western Blot Analysis 8 7.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8 Glycon Measurement In Mouse Quadriceps 9 8.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 8.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 8.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 8.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1 Culturing And Purifica- tion of Plasmid From Bac- terial Cells A liquid bacterial medium containing Escherichia coli was used to culture and purify two plasmids: pGFP and pGAA. Liquid bacterial cultures gen- erally produce large yields of plasmid in relatively short periods of time. Subsequent purification of collected plasmids involved bacterial lysis, plas- mid extraction and purification through filtering. The purified plasmids were intended for use in subsequent cellular transfection and restriction digestions. 1.1 Methodology A half-litre solution of 2YT medium was prepared as the primary medium for the bacterial culture. 8g of tryptone, 5g of yeast extract and 2.5mg of sodium chloride were combined with deionised water. The resulting medium was autoclaved at 121 °C for twenty minutes to sterilise it. Following autoclaving, the medium was allowed to cool down to an appropriate temperature and was subsequently inoculated with 5mL of liq- uid LB medium containing 1mL of E. coli bac- teria and the necessary antibiotics. The inoc- ulated solution was then subjected to shaking at 37 °C overnight to promote cellular division. The shaking process served to ensure oxygena- tion and the even distribution of bacteria within the medium. The plasmid was extracted from the bacterial cells through cellular lysis. The bacterial cell suspension was subjected to centrifugation at 500rpm for five minutes in order to condense the suspended cells into a pellet. The supernatant was removed, and the cells were lysed open by adding 13mL of P1 resuspension buffer and P2 lysis buffer. The solution was gently shaken for three to five minutes to ensure adequate bacterial lysis without causing damage to the plasmid. Following lysing, 13mL of P3 neutralisation buffer was added to neutralise the lysing effect of P2, en- suring the genomic integrity of the plasmid and preventing degradation. The solution was re- centrifuged at 5,000rpm for sixteen minutes to separate the bacterial debris and cellular com- ponents from the plasmid, which remained sus- pended in the supernatant. The supernatant was collected and the cellular debris was discarded. Plasmid extraction was conducted using Qiagen Tip-500 columns. 10mL of QBT equilibration buffer was passed through each column to ensure optimal binding conditions for the plasmid. The supernatant containing the plasmid was poured through. The plasmid selectively binds to the col- umn, allowing impurities to flow through. Resid- ual impurities was removed by washing the col- umn with 30mL of QC wash buffer, and the plas- mid was released from the column matrix using 15mL of QN elution buffer. To encourage precipitation of the plasmid DNA, the eluate solution was combined with 10.7mL of isopropanol and centrifuged at 13,000rpm for fifteen minutes, resulting in the DNA pelleting at the bottom. The supernatant was discarded, and the pellet was washed with 70% ethanol and briefly centrifuged to further remove any impu- rities. The DNA was re-dissolved in 1mL of TE buffer and was stored at -20 °C 1.2 Measured DNA Concentration DNA concentration was assessed through three trials, with each trial utilising 1 μ of DNA dissolved in TE buffer. The average measured concentra- tion for pGFP was 352ng/ μ L and 611ng/ μ L for pGAA. 2 Restriction Digestion of pGFP And pGAA A restriction digestion was performed to verify the type and purity of the collected plasmid. Three specific restriction endonucleases were used for each plasmid: PvuI (recognises CGATCG), HindIII (recognises AAGCTT) and EcoRI (recog- nises GAATTC) for pGFP (Figure 1a), and SacI (recognises GAGCTC), BamHI (recognises GGATCC) and MluI (recognises ACGCGT) for pGAA (Figure 2a). Agarose gel electrophoresis was used to produce an imageable product. 2.1 Methodology Plasmids cultured from E. coli bacteria were col- lected and thawed on ice. Three microtubes were prepared for the subsequent restriction enzyme digestion. Each microtube contained 16 μ L of wa- ter, 2 μ L of NEB buffer, 1 μ L of the specific plas- mid sample (pGFP and pGAA) and 1 μ L of the corresponding restriction enzyme. To ensure proper mixing, the microtubes were briefly centrifuged and placed in a water bath maintained at 37 °C for two hours to allow the restriction digestion process to proceed. A 1% agarose gel consisting of 0.25g of agarose and 25mL of C.5x tris-acetate-EDTA (TAE) buffer was prepared for subsequent gel elec- trophoresis. The agarose was combined with the 1 TAE buffer and heated until the agarose was com- pletely dissolved. The contents were allowed to cool for five minutes, and 10 μ L of GelRed fluo- rescent dye was added to the solution. GelRed was chosen for its ability to bind to DNA and rel- atively low toxicity. The gel solution was poured into a casting tray, and a comb was inserted at one end to create loading wells for the plasmid samples. Following gel solidification, the gel was trans- ferred to an electrophoresis box and was covered with C.5x TAE buffer. 4 μ L of loading buffer with a blue loading dye was added to each re- action sample to serve as a visual indicator of the progress of electrophoresis. 10 μ L of each reac- tion sample was loaded into a precast well, and the electrophoresis box was connected to a 100V power supply and was allowed to run for one hour. Following electrophoresis, the gel was transferred to a gel imaging system. 2.2 Results Agarose gel electrophoresis revealed the presence of two plasmids, pGFP and pGAA, as evidenced by the distinct bands observed in Figure 1b and Figure 2b. For all six restriction enzymes, the ob- served minor and major cut lengths corresponded with the expected theoretical lengths consistent with pGFP and pGAA (Table 1). Enzyme Minor Cut Major Cut Observed Minor Cut Observed Major Cut PvuI 2,329bp 9,306bp 2,750bp ± 500bp 9,500bp ± 500bp HindIII 2,106bp 9,529bp 2,750bp ± 500bp 9,500bp ± 500bp EcoRI 1,739bp 9,896bp 1,750bp ± 250bp 9,500bp ± 500bp SacI 924bp 10,711bp 1,000bp ± 100bp 10,000bp ± 500bp BamHI 2,613bp 9,022bp 2,650bp ± 150bp 9,000bp ± 500bp MluI 1,797bp 9,838bp 1,800bp ± 200bp 9,500bp ± 500bp Table 1: Restriction digestion results of pGFP and pGAA using six enzymes. (a) pGAA plasmid with denoted cutting points for restriction digestion. (b) Imaging of digested pGFP samples. Figure 1: Restriction digestion of pGFP plasmid. 2 (a) pGFP plasmid with denoted cutting points for restriction digestion. (b) Imaging of digested pGAA samples. Figure 2: Restriction digestion of pGAA plasmid. 3 pGFP And pGAA Trans- fection of COS-7 Cells COS-7 cells were transfected with plasmids en- coding green fluorescent protein (pGFP) and the synthesis of α -glucosidase (pGAA) in order to in- vestigate green fluorescent protein (GFP) expres- sion and α -glucosidase (GAA) activity. One plate of COS-7 cells (the primary plate) was subcul- tured into five. Two plates were each transfected with pGFP and pGAA using the calcium phos- phate method, and one was left untransfected as a control. The calcium phosphate transfec- tion method was chosen over electroporation and lipofection due to its relative ease of successful implementation. 3.1 Methodology To minimise the risk of contamination, the sub- culturing and transfection process were con- ducted under a laminar flow hood. A specific timing protocol was followed, where transfection took place twenty-four hours after subculturing. The time gap allowed the cells to undergo ade- quate division for efficient transfection. Three primary plates of COS-7 cells were subcul- tured into fifteen separate plates. The medium from each original plate was removed, and the cells were rinsed with 6mL of phosphate-buffered saline (PBS), which was then discarded. 1mL of trypsin-EDTA was added, and the plates were in- cubated at 37 °C for five minutes to facilitate cel- lular detachment. Following incubation, 9mL of cell culture medium was added to each plate to provide a suitable environment for cell growth. Each of the subcultures received 8mL of cell cul- ture medium along with 2mL of the contents from each original plate. To enable transfection in each of the subcul- tures, a transfection mix was prepared consisting of calcium, phosphate and the respective plas- mids. The pGFP-containing transfection mix consisted of 405.5 μ L of water, 62.5 μ L of 2M calcium chloride, 32 μ g of pGFP plasmid and 500 μ L of HEPES phosphate solution, which were added sequentially. The pGAA-containing trans- fection mix consisted of 393.5 μ L, 62.5 μ L of 2M calcium chloride, 44 μ L of pGAA plasmid and 500 μ L of HEPES phosphate solution. The result- ing plasmid concentrations were 0.311 μ g/ μ L and 0.226 μ g/ μ L for pGFP and pGAA respectively. Six plates were transfectd with pGFP. 5 μ g of pGFP was introduced to three plates while the other three plates received 0.5 μ g of plasmid. Six plates were transfected with 5 μ g of pGAA. To ensure an even distribution of the transfec- tion mixture, all transfected plates were gently swirled, and all plates were incubated at 37 °C 3 4 pGFP Expression pGFP is expressed through the synthesis of green fluorescent proteins (GFP). GFP is widely used to genetically encode fluorescent markers in cells. One day after pGFP was transfected to COS-7 cells, magnified UV imaging was done verify the presence of GFP. 4.1 Methodology Six plates of COS-7 cells transfected with pGFP were imaged one day after transfection to verify the expression of pGFP through the presence of GFP. 4.2 Results A positive correlation was observed between the quantity of transfected pGFP and the corre- sponding fluorescence intensity observed during imaging. Cells transfected with 5 μ g of pGFP had greater levels of gene expression than cells trans- fected with 0.5 μ g of pGFP. 4.3 Discussion The transfection of COS-7 cells with pGFP fol- lowed by the subsequent expression and visuali- sation of GFP served as a proof of concept in il- lustrating the ability of GFP to serve as a genetic marker for monitoring gene expression and facil- itating cellular imaging. In practise, GFP hosts additional applications in the studies of protein localisation, cellular functions, host-pathogen in- teraction and biotechnology, though such appli- cations were not considered nor demonstrated in the proof of concept. (a) Cells transfected with 5 μ g pGFP under 2x magnification. (b) Cells transfected with 0.5 μ g pGFP under 2x magnification. (c) Cells transfected with 5 μ g pGFP under 20x magnification. (d) Cells transfected with 0.5 μ g pGFP under 20x magnificationn. Figure 3: Expression of GFP in COS-7 cells one day after transfection. 4 5 Assessing GAA Expression Through α -Glucosidase Activity And BCA Assays In COS-7 and other mammalian cells, the GAA gene is expressed through the synthesis of the α - glucosidase enzyme. The α -glucosidase enzyme is primarily used to break down glycogen into glu- cose. Mutations in the GAA gene may result in a deficiency of α -glucosidase, leading to various diseases including Glycogen Storage Disease Type II (Pompe Disease). To assess GAA expression, COS-7 cells were transfected with pGAA encod- ing the GAA gene. Following transfection, the ac- tivity of the α -glucosidase enzyme was measured for cells on six transfected plates and compared to cells on three non-transfected controls. To fur- ther quantify GAA expression, a bicinchoninic acid (BCA) assay was performed to obtain total protein concentrations of cells from each plate, which was used to assess the overall expression of GAA. 5.1 Methodology To analyse α -glucosidase enzyme activity and protein concentrations, six plates each transfected with 5 μ g of pGAA and three non-transfected plates were utilised. The nine plates were di- vided into three group, each containing one non- transfected (NT) control plate and two trans- fected (T) plates. To begin the cellular extraction process, COS-7 cells from each plate were detached and trans- ferred to microtubes. The medium in the plates was vacuum-removed, and 5mL of phosphate- buffered saline (PBS) bufer was added to wash the cells and discarded. 1mL of clean PBS buffer was re-introduced, and the cells were gen- tly scraped off and transferred to microtubes on ice. Following extraction, the microtubes were cen- trifuged at 5,000rpm for five minutes to pellet the cells. The supernatant was removed and 500 μ L of water was added to resuspend the cells. To lyse the cells, three rounds of sonication was per- formed, each lasting for fifteen seconds. Following sonication, a second round of centrifu- gation was performed at 12,000rpm to separate cell debris from the desired cellular components. The supernatant was collected and the pellet was discarded. To measure α -glucosidase activity, a quantitative enzyme assay was performed. 20 μ L of substrate specific to the α -glucosidase enzyme and 10 μ L of each test sample was added to wells on a 96-well plate. The process was duplicated, and the en- zymatic reaction was allowed to proceed for one hour at 37 °C . Subsequently, 130 μ L of high-pH car- bonate stop buffer was added to each well to ter- minate enzymatic activity, and a microtitre plate reader was used to measure α -glucosidase activ- ity. Protein concentrations were quantified using a bicinchoninic acid (BCA) assay. 20 μ L of each test sample was diluted tenfold in water. Premade reagents A and B were combined in a 50:1 ratio to form a mixture that synthesises a coloured com- plex with proteins. Subsequently, 10 μ L of each diluted test sample and 1mL of the reagent solu- tion were combined in a microtube. The microtubes were set in a 55 °C water bath for thirty minutes to allow for the synthesis of coloured complexes. Following incubation, ab- sorbance was measured at a wavelength of 652nm using a spectrophotometer. The 652nm wave- length is specific to the colour of the protein com- plex produced by the BCA reaction. 5.2 Results COS-7 cells transfected with pGAA encoding the GAA gene exhibited elevated α -glucosidase en- zyme activity and specific α -glucosidase activity. A BCA assay revealed protein concentrations re- mained relatively stable between transfected and non-transfected cells (Table 2). Transfected cells from Group 1 and Group 3 ex- hibited α -glucosidase activity three-to-six times greater than cells originating from the non- transfected control plate. No meaningful corre- lation in protein concentrations was observed in transfected and non-transfected cells from Group 1 and Group 2. α -glucosidase specific activity was higher in cells in Group 1 transfected with pGAA than cells originating from the non-transfected control plate. Due to discrepancies or anomalies in the data, some measurements obtained from Group 2 and Group 3 were excluded from analysis. Excluded data highlighted in red in Table 2. Further anal- ysis on data reliability is presented in the Discus- sion. 5 Protein (BCA) α -Glucosidase Activity λ mg/mL Raw Readings (U/mL) Mean (U/mL) Specific Activity (U/mg) NT 0.192 0.199277 50.0735 50.0159 50.0447 251.1 T 0.112 0.116245 297.998 274.062 286.03 2460.6 Group 1 T 0.146 0.151533 286.967 271.424 279.1955 1842.5 NT 0.127 0.131813 2.71723 18.4669 10.59207 80.4 T 0.153 0.158799 22.6275 5.50049 14.064 88.6 Group 2 T 0.094 0.097563 2.47406 2.47406 25.4 NT 0.06 0.062274 32.9754 31.1309 32.05315 514.7 T 0.019 0.01972 200.447 198.848 199.6475 10124.1 Group 3 T 0.023 0.023872 97.0845 100.665 98.87475 4141.9 Table 2: α -glucosidase activity and protein concentration measurements. Group 1 Group 2 Group 3 0 0.05 0.1 0.15 0.2 (a) Absorbance readings at λ =562nm. Group 1 Group 2 Group 3 0 0.05 0.1 0.15 0.2 0.25 Protein Concentration (mg/mL) (b) Measured protein concentration. Figure 4: Measured λ and protein concentrations of non-transfected (blue) and transfected (red) cells. Group 1 Group 2 Group 3 0 100 200 300 Mean Enzyme Activity (U/mL) (a) Mean α -glucosidase activity. Group 1 Group 2 Group 3 10 0 10 1 10 2 10 3 10 4 Specific Enzyme Activity (U/mg) (b) Specific α -glucosidase activity. Figure 5: Measured α -glucosidase activity for non-transfected (blue) and transfected (red) cells. 5.3 Discussion Due to concerns regarding the reliability and ac- curacy of certain portions of the collected data, data from Group 2 and Group 3 were excluded from the analysis of α -glucosidase activity and protein concentrations respectfully. Based on the analysis of enzyme activity data 6 from Group 1 and Group 3, it was observed that transfected cells demonstrated a statistically sig- nificant increase in α -glucosidase activity. Trans- fected cells from Group 1 exhibited an average increase of 222.7U/mL, which was 4.45-fold in- crease in activity when compared to the control. Similarly, transfected cells from Group 3 showed an average increase of 117.2U/mL, representing a 3.66-fold increase compared to the control. In contrast, transfected cells from Group 2 exhib- ited an average decrease of 2.3U/mL in activ- ity representing a 28% decrease. Furthermore, α -glucosidase activity of transfected cells from Group 2 was not determined to be statistically significant, and as a result, the data from Group 2 was excluded from the analysis of enzyme activ- ity and specific activity. Analysis in α -glucosidase activity instead focused on measurements from Group 1 and Group 3, where enzyme activity trends were more concretely established. With regards to the BCA assay, all three Groups showed no meaningful increase in absorbance ( λ ) at 562nm or protein concentrations between transfected and non-transfected cells. However, it was observed that cells from Group 3 had sig- nificantly lower measured absorbance and pro- tein concentrations than cells from Group 1 and Group 2. In Group 1, the average absorbance and protein concentrations of the transfected cells was measured at 0.129 and 0.134mg/mL respec- tively. Similarly, within the same order of mag- nitude, transfected cells from Group 2 exhibited and average absorbance of 0.124 and an aver- age protein concentration of 0.128mg/mL. Trans- fected cells from Group 3 exhibited average ab- sorbance of 0.021 and an average protein concen- tration 0.021796mg/mL, approximately 0.78 or- ders of magnitude lower than that observed in Group 1 and Group 2. Due to the substantial de- viation in absorbance and protein concentrations, data from Group 3 was excluded from the analy- sis of protein concentrations and specific activity, and conclusions drawn from the BCA assay relied upon data collected from cells from Group 1 and Group 2. To identify the specific error or errors resulting in the observed corrupted data, a careful review of experimental procedures, sample handling and reagant preparation would be necessary. A trans- fection failure is unlikely to be the primary rea- son for the corrupted data as no group exhibited corrupted readings of protein concentration and α -glucosidase activity. As a transfection failure would likely impact both sets of data, it sug- gests that errors during the preparation of en- zyme activity measurement and BCA assay are more probable causes for the corrupted data. Similarly, as a restriction digestion of the cul- tured plasmid confirmed the presence and purity of pGAA, it is unlikely the corrupted data was a result from impure plasmids. Potential sources of error may include the improper allocation of test samples, impurities present in the stop buffer used to measure enzyme activity or impurities present within the reagents used during the BCA assay. Regardless, it is exceeding unlikely to iso- late the exact error. 5.4 Conclusion COS-7 cells transfected with pGAA encoding the GAA gene exhibited elevated α -glucosidase en- zyme activity and specific α -glucosidase activ- ity. A BCA assay revealed Prontein concentra- tions Ignoring corrupted data, enzyme activity was largely elevated in COS-7 cells transfected with pGAA, demonstrating the expression of the GAA gene through the synthesis of α -glucosidase. The combination of a BCA assay and an enzyme activity measurement is highly relevant in the confirmation of Glycogen Storage Disease Type II (GSDII). GSDII is characterised by a muta- tion on the GAA gene resulting in a deficiency of α -glucosidase activity, resulting in the accu- mulation of glycogen in muscle tissue. Enzyme activity measurements and BCA assays provide a direct tool to detect abnormal α -glucosidase pres- ence and assess GAA expression in cells or tissues affected by GSDII. The enzyme activity measurement provides in- sights into the functional status of the enzyme, quantifying the ability for glycogen to be bro- ken down into glucose. Enzyme activity mea- surements thus can determine the extent of α - glucosidase deficiency and dysfunction in individ- uals affected with GSDII. Enzyme activity mea- surements combined with a BCA assay may pro- vide a more comprehensive analysis useful in diag- nosing GSDII, assessing disease severity, monitor- ing disease progression and evaluating the efficacy of treatments. In particular, BCA assays and en- zyme activity measurements are useful in devel- oping and evaluating enzyme replacement thera- pies. Such procedures can assess the expression of theraputic GAA proteins, evaluate the efficacy of ERT and monitor the response to treatment. 6 Genotyping of Mouse Tails Mice used in research are often genetically modi- fied or bred for specific genetic traits. The geno- typing process provides the ability to identify specific genetic markers, mutations or transgenes 7 present in a mice’s genome. In this study, geno- typing was performed to determine the genetic composition of three mice to determine whether the offspring of two heterozygous transgenic mice was wild-type or knock-out. 6.1 Methodology Genotyping of mice tails was conducted using four samples: p1327, p1330, p1343, and p1344. The tails were placed in a solution of 180 μ L of ly- sis buffer and 1 μ L of Proteinase K. The contents were then incubated in a water bath set at 55 °C overnight to promote cellular lysis and the release of genomic DNA. Following cellular lysis, the contents were heated to 85 °C to deactivate the Proteinase K enzyme. The DNA was precipitated by adding 120 μ L of 5M sodium chloride. The resulting mixture was then centrifuged at 11,000 rpm for six minutes to pellet the DNA. The supernatant was removed, and 400 μ L of ethyl alcohol was added to wash and purify the DNA pellet. The mixture was recen- trifuged at 14,000rpm for five-and-a-half minutes, the supernatant was removed and teh pellet was extracted and air-dried for twenty minutes. Following air-drying, the DNA pellet placed in 200 μ L of TE buffer and was set in a 55 °C water bath to resuspend the DNA. After resuspension, the DNA was stored at 4 ° C. In preparation for a polymerase chain reaction (PCR), a master mix consisting of 16 μ L of deionised water, 25 μ L of 2X Taq DNA poly- merase mix and 1 μ L of a combination of a forward primer, of which there were two options available and a reverse primer. 1 μ L of the DNA sample alongside 9 μ L of master mix were combined in a PCR tube. The PCR tubes were briefly spun to ensure proper mixing and placed in a thermocy- cler PCR machine to undergo the PCR process. The PCR process consisted of an initial denatur- ing step at 94 °C for three minutes and 35 am- plification cycles. Each cycle consisted of a de- naturing step at 94 °C , an annealing step at 56 °C and an extension step at 72 °C . Each amplifica- tion step lasted for twenty seconds, resulting in a total PCR process time of one-and-a-half hours. Following the amplification cycles, the PCR prod- ucts were held at 10 °C indefinitely. The PCR products were subjected to agarose gel electrophoresis. A 1.5% agarose gel consisting of 1.2g of agarose and 80 mL of 0.5X tris-acetate- EDTA (TAE) buffer. The mixture was heated to allow the agarose to dissolve completely and sub- sequently cooled for five minutes. 35 μ L of GelRed fluorescent dye was added to the gel solution and the solution was poured into a casting tray. A comb was inserted into the gel solution to create loading wells for the PCR products. Following solidification, the comb was removed and the gel was transferred to an electrophoresis box and covered with C.5X TAE buffer. 12 μ L of 1kb DNA ladder was added to each well at the ends of the gel, and 6 μ L of each PCR product was placed in between. The gel was subjected to a 120V electric field for one hundred minutes and subsequently imaged. 7 Western Blot Analysis The Western blot technique provides the ability to detect, identify and quantify proteins of inter- est within complex biological samples. Western blots are regarded for their exceptional sensitivity and specificity, enabling the accurate detection of proteins present in low concentrations. Such ben- efits are achieved through the combination of gel electrophoresis to separate proteins by molecular weights and antigenic properties, and the utilisa- tion of protein-specific binding antibodies. The Western blot technique is a multi-step process involving protein extraction, gel elec- trophoresis, protein transfer to a solid membrane and antibody incubation. Western blots can be further enhanced with modifications such as mul- tiplexing to enable the simultaneous detection of multiple proteins within a single experiment. This study employs a generic Western blot tech- nique. 7.1 Methodology The core steps of a Western blot assay in- clude protein extraction, polyacrylamide gel elec- trophoresis, protein transfer, blocking and two rounds (primary and secondary) of antibody in- cubation. Proteins are extracted from a biologi- cal sample, and polyacrylamide gel electrophore- sis is employed to separate proteins to different locations on the gel matrix based on their re- spective molecular weights. Proteins are subse- quently transferred onto a nitrocellulose mem- brane to immobilise further protein movement and to enable the subsequent antibody incuba- tion process. Milk is used as a blocking agent to prevent nonspecific antibody binding, improving the specificity of antibody detection. The mem- brane is incubated with a primary antibody that binds to the target protein and a secondary an- tibody conjugated to an enzyme for subsequent imaging purposes. Test solutions were synthesised by combining 30 μ L of each sample of interest with 10 μ L of 8 4X SDS loading buffer. The test solutions were mixed and boiled for three minutes to denature the proteins. A pre-formed small polyacrylamide gel was covered with loading buffer, and the comb was removed. 15 μ L of each test solution was loaded into each well of the gel. To track pro- tein migration and estimate their sizes, molecular weight markers were placed on both ends of the gel. Gel electrophoresis was conducted at 100V for one hour. Larger gels require higher volt- ages and longer electrophoresis times: 150V-250V for at least two hours, or 35V-40V overnight. Figure 6: Western blot transfer sandwich setup. Following gel electrophoresis, proteins on the polyacrylamide gel were transferred onto a ni- trocellulose membrane to immobilise the proteins and to enable antibody binding and subsequent detection. A transfer sandwich consisting of the polyacrylamide gel, a nitrocellulose membrane, filter paper and absorbant pads (Figure 6) was prepared. Transfer buffer, typically containing tris and glycine, was added to the transfer sand- wich to maintain a suitable pH and ionic strength for the efficient transfer of proteins. The sand- wich assembly was placed inside a gel box filled with water. A constant current of 250mA was passed through the gel box for one hour to facili- tate the protein transfer process. Following protein transfer, the nitrocellulose membrane containing the transferred proteins was incubated with a primary antibody solu- tion consisting of deionised water, 1% milk in TBST (tris-buffered saline with Tween-20) and mouse anti-acid α -glucosidase (anti-GAA) anti- body. The addition of milk in TBST improves the specificity of the Western blot by preventing nonspecific binding. The nitrocellulose membrane was immersed in the primary antibody solution and shaken overnight in a cold room to ensure the thorough expore of the proteins to the primary antibody. Sub- sequently, the membrane was removed from the primary antibody solution and washed by shak- ing it in fresh TBST for five minutes three times to remove any excess of non-bound primary anti- body. Subsequent to the washing steps, a sec- ondary antibody incubation solution was pre- pared consisting of TBST alongside 1:10,000- diluted sheep anti-mouse IgG antibody conju- gated with horseradish peroxidase (HRP). The nitrocellulose membrane was transferred into the secondary antibody incubation solution, shaken for one hour and washed in an identical manner. The presence of HRP in the secondary antibody enabled subsequent imaging. 8 Glycon Measurement In Mouse Quadriceps Glycogen is a carbohydrate used for energy stor- age stored in skeletal muscle of animals. Glyco- gen is mainly used during energy-intensive activ- ities, and plays a key role in regulating muscle metabolism and contractile function. The quadriceps are a group of muscles located in the front of the thigh. As the largest mus- cles in the leg, the quadriceps play an im- portant role in movement and locomotion, and thus have high metabolic demand during exer- cise and movement. The quadricepts are capa- ble of rapidly depleting and replenishing glyco- gen stores, thus, studying glycogen content specif- ically in the quadriceps muscle tissue of mice can yield useful information regarding the muscular energy storage capacity and metabolism of the leg muscles. 8.1 Methodology Glycogen measurements were conducted by mea- suring the quantity of NADH present in each bio- logical sample after the introduction of amyloglu- cosidase. Amyloglucosidase catalyses the hydrol- ysis of glycogen, converting it into glucose. When in the presence of glucose, nicotinamide adenine dinucleotide (NAD) is reduced to NADH. The reduction is accompanied by a colour change as NADH is strongly absorbs light with a wavelength of 420nm. The absorbance at 420nm directly cor- relates to the quantity of NADH, which correlates with the initial amount of glycogen present. Two quadriceps were collected from each of the three test mice (A366, A370, A371), as well as a control mouse (W851). Each quadricep was weighed, and each quadricep was placed in a tube containing 20 μ L of water per mg of tissue. Each quadricep was homogenised to release its cellular contents for subsequent analysis. Following homogenisation, the samples were cen- trifuged at 18,200rpm for five minutes. The resulting supernatant was transferred to micro- tubes, and the cellular debris was discarded. 9 Two test solutions, one positive and one nega- tive, were prepared for each test sample. 55 μ L of water and 20 μ L of lysate were added to each test solution, and the contents were subsequently boiled for three minutes. 25 μ L of amyloglucosi- dase diluted 1:50 in KAC buffer was added to the positive test solution, and 25 μ L of KAC buffer without amyloglucosidase was added to the neg- ative test solution. All test solutions were briefly spun and incubated in a 37 °C water bath for ninety minutes to allow the enzymatic reaction to proceed. Following incubation, the test solutions were re-boiled for three minutes to terminate the enzymatic reaction by denaturing the amyloglu- cosidase present and briefly spun. 30 μ L of test solution was transferred into a new microtube, and 1mL of a glucose reagent was added to each test solution to initiate the colourimetric reaction. The reaction was allowed to proceed for ten min- utes, and the absorbance of the reaction mixture was measured at 420nm using a spectrophotome- ter. Quad 1 Quad 2 0 0.01 0.02 0.03 Glycogen Concengration ( μ mol/mg) (a) W851. Quad 1 Quad 2 0 0.01 0.02 0.03 Glycogen Concengration ( μ mol/mg) (b) A366. Quad 1 Quad 2 0 0.01 0.02 0.03 Glycogen Concengration ( μ mol/mg) (c) A370. Quad 1 Quad 2 0 0.01 0.02 0.03 Glycogen Concengration ( μ mol/mg) (d) A371. Figure 7: Measured glycogen concentration in control mice (W851) and test mice quadriceps treated with (blue) and without (red) amyloglucosidase. 8.2 Results The present study aimed to investigate the glyco- gen concentrations in the quadriceps muscles of mice with specific genetic variations. Three test mice with the identification codes A366, A370, and A371 were compared to a control group of mice (W851). Glycogen concentrations from quadricep samples reacted with amyloglucosidase was subtracted from background glycogen con- centrations (no amyloglucosidase) to calculate the net glycogen concentration variation (NGCV) (Figure 8). 10 8.3 Discussion W851 A366 A370 A371 0 20 40 60 80 Glycogen Concentration ( μ mol/g) Figure 8: NGCV in control mice (W851) and test mice in two quadricep samples. All three test mice exhibited greater NGCV than the control. The respective NGCV and mean absolute deviations in A366, A370 and A371 were 63.8333 μ mol/mg ± 5.5 μ mol/mg, 53.1667 μ mol/mg ± 17.5 μ mol/mg and 61.3333 μ mol/mg ± 1.3333 μ mol/mg, represent- ing over an order of magnitude’s increase when compared to the control sample (1.8333 μ mol/mg ± 0.1667 μ mol/mg). The results of this study demonstrate that certain genetic variations present in mice are expressed through significantly higher glycogen concentrations in their muscles. The observed increase indicates a poten- tial alteration in the glycogen synthesis, metabolism or storage process. As glyco- gen primarily serves as an energy reserve, higher glycogen levels in these mice may suggest improved muscle performance or en- durance. The limited sample size (three test mice and one control) and purpose reduces the statistical and scientific power of these findings. The study was conducted as a demonstration of scientific methods used to determine glycogen concentra- tions in animals, and did not consider potential effects. It would be valuable to expand the scope of glycogen research beyond storage alone to pro- vide a more comprehensive understanding of the physiological significance these genetic variations impart on their hosts. 8.4 Conclusion This study provided insight as to the method used to measure glycogen concentrations within ani- mal muscle tissue. Genetic variations imparted on three test mice may have affected their abilities to synthesise or metabolise glycogen, resulting in the higher NGCV. As this study did not consider the physiological aspects nor made an attempt to identify the specific genetic variance observed, further research should be conducted with larger sample sizes alongside genetic and functional as- sessments. Understanding the molecular factors involved in glycogen regulation may enable fur- ther development of treatments against glycogen storage diseases and other metabolic disorders and enhance muscle performance and retention. 11