9:03CQ:E Assignment detailsFunctional Genomics and Proteomics (2250) DescriptionPlease see the prac...

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9:03CQ:E Assignment detailsFunctional Genomics and Proteomics (2250) DescriptionPlease see the prac notes for what you will do in thepracticals. Our research lab specialises in medicinalplants for chemotherapy applications. This year, you canjoin us in finding out if our he can reduce skin cancers,specifically melanoma : download prac notes for group 1here. Due to the disappointing results from group 1, |have modified the prac notes for group 2- downloadhere.Some interesting facts about skin cancers to get you inthe zone and thinking about the impact of this researchin real life:aoe SKINCANCERFACTS 9,3OVER—~WHAT YOU NEED TO KNOW-20 PER DAY AUSTRALIANS WILL BEARE TREATED FOR SKIN CANCER DIAGNOSED WITHA hs. aii ih SKIN CANCEREE . oo BY THE AGE OF 70AUSTRALIA'S MOST MORE THAN. CASES OFCOMMON 12,500 itionC A N C k R . . ARE DIAGNOSED IN AUSTRALIA EACH YEARasa. Te MEN ARE MORE THANMELANOMAISS MORE COMMON2000 ENG SimI DEATHS PER YEARo> He DONT BE A STAT. \ SLIP, SLOP, SLAP, SEEK & SLIDE...N AND SPOTSubmit assignmentan BH OB NO =DDashboard Calendar To-do Notifications Inbox   | have also included a short video of loading the samplesin the qPCR machine to help you understand the process | will load up your qPCR results so you can write up youeport. Keep an eye on this page as it will be updatedonce | get the results.Results for group 1 download here! (melt curves notincluded)Results for group 2 !There will also be a tutorial in collaborate ultra on 9thSeptember @ 1:30pm to go through your calculations buthave an attempt first to optimise your learning.Submit assignmentan BH OB NO =DDashboard Calendar To-do Notifications Inbox  BIOL2267 – Functional Genomics & Proteomicsiol2267 – functional genomics & proteomicsBIOL2267 – Functional Genomics & ProteomicsQuantitative PCR (real-time PCR)IntroductionYou have been hired as a medical research assistant in our lab to investigate medicinal plants to augment cancer development. In this practical, you will use molecular biology to quantify the effect of an exotic he that reduces melanoma on human cells using QPCR. This he acts on the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway that causes downstream activation of inflammatory and antioxidant derivatives including Tumour necrosis factor alpha (TNF-(), Nuclear factor erythroid 2-related factor 2 (Nrf2) compared to the housekeep gene GAPDH (Figure 1). Figure 1. Signalling pathways by active phytochemicals to augment cancer chemotherapy and immunotherapy (https:www.frontiersin.org/articles/10.3389/fonc XXXXXXXXXX/full). TLRs=Toll-like receptors. Learning outcomes· To accurately and consistently dispense small volumes with a micropipette.· To create a standard curve for amplification efficiency for qPCR. · To perform qPCR on untreated and treated samples.· To analyse qPCR performance, melt curves and co-efficiency cohorts.· To write a scientific report on the benefits of our exotic he for chemotherapy.InstructionsA. Calculation of cDNA to usecDNA were extracted and diluted to 1 (g/(L for you to use. Work in groups and set up the experiment as a class. The Gene of Interest (GOI) primers change with stimulation and treatment (=NFkB, TNFa, Nrf, p53). Housekeeping (HK) primers are constitutively expressed, always on and shouldn’t change (=GAPDH).1. You will form 2 groups.2. Group 1 will setup the standard curve with different primers ± xng cDNA and controls3. Group 2 will setup the cDNA samples with different primers and 1ng cDNA.B. Setup of the standard curve – this has already been done by group 11. Group 1 (8 students) will set up the standard curve using the 4 different primers with different cDNA concentrations of untreated H cells (labelled as HU). Include a no template control (NTC=0.0ng cDNA). Refer to table 1.Table 1. Setup of the standard curve and controls.    Student     Well 1    Well 2    Well 3    Well 4    A    Standard GAPDH0ng HU    Standard GAPDH1ng HU    Standard GAPDH2ng HU    Standard GAPDH 4ng HU    B    Standard GAPDH0ng HU    Standard GAPDH1ng HU    Standard GAPDH2ng HU    Standard GAPDH 4ng HU    C    Standard NFkB0ng HU    Standard NFkB1ng HU    Standard NFkB2ng HU    Standard NFkB4ng HU    D    Standard NFkB0ng HU    Standard NFkB1ng HU    Standard NFkB2ng HU    Standard NFkB4ng HU    E    Standard TNFa0ng HU    Standard TNFa1ng HU    Standard TNFa2ng HU    Standard TNFa4ng HU    F    Standard TNFa0ng HU    Standard TNFa1ng HU    Standard TNFa2ng HU    Standard TNFa4ng HU    G    Standard Nrf0ng HU    Standard Nrf1ng HU    Standard Nrf2ng HU    Standard Nrf4ng HU    H    Standard Nrf0ng HU    Standard Nrf1ng HU    Standard Nrf2ng HU    Standard Nrf4ng HU2. Calculate what will be needed to create your mastermix (table 2). Why do you need a mastermix? Make your mastermix containing your qPCR mix, primers (forward and reverse) multiplied by 5 (fill in table 2). Double check your calculations! Remember you need enough for 4 samples so there should be plenty left over! Too little or too much and what happens? Table 2. Constituents for a qPCR standards (Sensimix Sy no rox).    Component (label)    (L per reaction    [Final]    Mastermix for standards(L (x5)    qPCR mix (Q)    6    1x        Forward Primer (gene F)    1    500nM        Reverse Primer (gene R)    1    500nM        DEPC wate    *    -    * (add separate)    cDNA (HU) (?ng)    *    -    * (add separate)    Total (l aliquoted per well    8    -        Total (l volume    12.5    -    *indicates variable amounts required to make the final total volume3. Wearing gloves, wipe down your bench area with 70% ethanol.4. Make the standards mastermix according to table 2 containing the qPCR mix and primers (forward and reverse) in an eppendorf. Mix by pipetting up and down gently.5. Grab a QPCR 4-well. Make note of where well 1 is by drawing a dot on the lid.6. Transfer 8uL of the mastermix to each well. 7. If you get to the last well and there is not enough, you have over pipetted and it’s game over! ☹8. Then add cDNA to the concentration required. Fill up to total volume with the DEPC water. 9. Close the lids. Remove air bules by flicking or tapping wells. Keep on ice and in darkness. 10. Your sample is now ready to run in a qPCR and you have finished the practical. 11. Wait for the rest of the class to load their PCR strip tubes. Note your group number and details of each person who pipetted what to write in your report.C. Setup of the cDNA samples.1. Group 2 will have 2 students in each group.2. Each group will set up the wells according to table 3 exactly. Table 3. Setup of the cDNA samples with and without 1mg/mL plant extract treatment (untreated=U, treated=T) for each of the different cell types (melanomas: C1, C32, D24, control H). Different primers are H    Group    Well 1    Well 2    Well 3    Well 4    Well 5    Well 6    Well 7    Well 8    C1 cellsA    Sample GAPDH 1ng cDNA untreated    Sample GAPDH1ng cDNA untreated    Sample GAPDH1ng cDNA untreated        Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated        B    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated        Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNAtreated        C    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated        Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated        D    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated        Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated        Group    Well 1    Well 2    Well 3    Well 4    Well 5    Well 6    Well 7    Well 8    C32 cellsE    Sample GAPDH1ng cDNA untreated    Sample GAPDH1ng cDNA untreated    Sample GAPDH1ng cDNA untreated        Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated        F    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated        Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNA treated        G    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated        Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated        H    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated        Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated        Group    Well 1    Well 2    Well 3    Well 4    Well 5    Well 6    Well 7    Well 8    D24 cellsI    Sample GAPDH1ng cDNA untreated    Sample GAPDH1ng cDNA untreated    Sample GAPDH1ng cDNA untreated        Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated        J    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated        Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNA treated        K    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated        Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated        L    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated        Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated        Group    Well 1    Well 2    Well 3    Well 4    Well 5    Well 6    Well 7    Well 8    H cellsM    Sample GAPDH1ng cDNA untreated    Sample GAPDH1ng cDNA untreated    Sample GAPDH1ng cDNA untreated        Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated    Sample GAPDH1ng cDNA treated        N    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated    Sample NFkB1ng cDNA untreated        Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNA treated    Sample NFkB1ng cDNA treated        O    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated    Sample TNFa 1ng cDNA untreated        Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated    Sample TNFa1ng cDNA treated        P    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated    Sample Nrf1ng cDNA untreated        Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated    Sample Nrf1ng cDNA treated    3. Create a mastermix. Calculate what will be needed to

Dr Shweta · Accepted Answer

1.Objective of the experiment: Quantification of the outcome of an exotic herb that decreases the melanoma on human cells with the help of QPCR.
2. Methodology: This experiment was performed by two groups: 
Group 1 made the standard curve using different primers ± xng cDNA and control while Group 2 used the cDNA samples with different primers and 1ng cDNA.
3. Results:
A) Group 1 members uses 4 different types of primers with different concentrations of cDNA of untreated H cells (HU) and a no template control (NTC=0.0ng cDNA).
The standard curve graph for all the primers is designed by plotting the Ct values with the logarithmic value of DNA copies present in per ml of the sample. The graph along with the slope and R2 value is shown as below:

9:03 CQ:E Assignment details Functional Genomics and Proteomics (2250) Description Please see the prac notes for what you will do in the practicals. Our research lab specialises in...

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