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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 medicinal plants for chemotherapy applications. This year, you can join us in finding out if our herb can reduce skin cancers, specifically melanoma : download prac notes for group 1 here. Due to the disappointing results from group 1, | have modified the prac notes for group 2- download here. Some interesting facts about skin cancers to get you in the zone and thinking about the impact of this research in real life: aoe SKINCANCERFACTS 9,3 OVER —~WHAT YOU NEED TO KNOW- 20 PER DAY AUSTRALIANS WILL BE ARE TREATED FOR SKIN CANCER DIAGNOSED WITH A hs. aii ih SKIN CANCER EE . oo BY THE AGE OF 70 AUSTRALIA'S MOST MORE THAN . CASES OF COMMON 12,500 ition C A N C k R . . ARE DIAGNOSED IN AUSTRALIA EACH YEAR asa. Te MEN ARE MORE THAN MELANOMAIS S MORE COMMON 2000 ENG Sim I DEATHS PER YEAR o> He DONT BE A STAT . \ SLIP, SLOP, SLAP, SEEK & SLIDE... N AND SPOT Submit assignment an BH OB NO =D Dashboard Calendar To-do Notifications Inbox | have also included a short video of loading the samples in the qPCR machine to help you understand the process | will load up your qPCR results so you can write up your report. Keep an eye on this page as it will be updated once | get the results. Results for group 1 download here! (melt curves not included) Results for group 2 ! There will also be a tutorial in collaborate ultra on 9th September @ 1:30pm to go through your calculations but have an attempt first to optimise your learning. Submit assignment an BH OB NO =D Dashboard Calendar To-do Notifications Inbox BIOL2267 – Functional Genomics & Proteomics biol2267 – functional genomics & proteomics BIOL2267 – Functional Genomics & Proteomics Quantitative PCR (real-time PCR) Introduction You 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 herb that reduces melanoma on human cells using QPCR. This herb 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.2022.834072/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 herb for chemotherapy. Instructions A. Calculation of cDNA to use cDNA 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 controls 3. 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 1 1. 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 GAPDH 0ng HU Standard GAPDH 1ng HU Standard GAPDH 2ng HU Standard GAPDH 4ng HU B Standard GAPDH 0ng HU Standard GAPDH 1ng HU Standard GAPDH 2ng HU Standard GAPDH 4ng HU C Standard NFkB 0ng HU Standard NFkB 1ng HU Standard NFkB 2ng HU Standard NFkB 4ng HU D Standard NFkB 0ng HU Standard NFkB 1ng HU Standard NFkB 2ng HU Standard NFkB 4ng HU E Standard TNFa 0ng HU Standard TNFa 1ng HU Standard TNFa 2ng HU Standard TNFa 4ng HU F Standard TNFa 0ng HU Standard TNFa 1ng HU Standard TNFa 2ng HU Standard TNFa 4ng HU G Standard Nrf 0ng HU Standard Nrf 1ng HU Standard Nrf 2ng HU Standard Nrf 4ng HU H Standard Nrf 0ng HU Standard Nrf 1ng HU Standard Nrf 2ng HU Standard Nrf 4ng HU 2. 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 Sybr 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 water * - * (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 volume 3. 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 bubbles 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 cells A Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated B Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated C Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated D Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated Group Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8 C32 cells E Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated F Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated G Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated H Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated Group Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8 D24 cells I Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated J Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated K Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated L Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated Group Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 Well 7 Well 8 H cells M Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA untreated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated Sample GAPDH 1ng cDNA treated N Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA untreated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated Sample NFkB 1ng cDNA treated O Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA untreated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated Sample TNFa 1ng cDNA treated P Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA untreated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated Sample Nrf 1ng cDNA treated 3. Create a mastermix. Calculate what will be needed to