Biology Lab Report

Anonymous
timer Asked: Dec 8th, 2017
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Question description

I have to write a lab report in 3 pages minimum about real time PCR demonstration. the lab report must be in 4 different sections, which are objective, background, results, and discussion. I am attaching the ppt as well as an Excel sheet of all students results including my result and I was student number 14. On the result section, we need calculate the concentration of an unknown sample using a Standard Curve. you can find more details in the attached ppt. In addition, I am attaching the instruction paper that show what each section should include.


Biology Lab Report
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BCHB 507/508 Laboratory Applications of Biotechnology Lab 999: Real Time PCR Demonstration TheChain PCR Reaction The Polymerase Reaction (PCR) pcr product plateau phase linear phase exponential phase 0 5 10 15 20 25 30 35 cycle # Regular PCR vs. qPCR Forward Primer  Reverse Primer  dNTPs  Thermophilic DNA  Polymerase  Template Forward Primer  Reverse Primer  dNTPs  Thermophilic DNA  Polymerase  Template   • But one more thing: – Fluorescent DNA dye How does dye work? How doesthe thefluorescent fluorescence dye work?  Ethidium Bromide and SyBr Green bind to dsDNA   emit light when illuminated with specific wavelength  Increase in PCR product leads to increase in fluorescence  Disadvantage: SyBr Green bind to all dsDNA products Intercalated and fluorescent SyBr Taq Polymerase Incoming SyBr http://www.sigmaaldrich.com/life-science/molecular-biology/pcr/quantitative-pcr/sybr-green-based-qpcr http://www.bbioo.com/experiment/12-770-3.html qPCR Applications     Gene expression analysis  Cancer research  Drug research Disease diagnosis and management  Viral quantification Food testing  Percent GMO food Animal and plant breeding  Gene copy number http://www.wonderwhizkids.com/index.php/bio-technology qPCR in Gene Expression Analysis BRCA1 Expression Profiling  BRCA1 is a gene involved in tumor suppression.  BRCA1 controls the expression of other genes.  In order to monitor level of expression of BRCA1, real-time PCR is used. BRCA1 http://slideplayer.com/slide/738141/ qPCR in Disease Treatment Example: HIV Treatment  Drug treatment for HIV infection often depends on monitoring the “viral load”  qPCR allows for direct measurement of the amount of the virus RNA in the patient  Measure amount of virus  adjust prescriptions http://medimoon.com/2012/10/fresh-lead-emerges-in-aids-vaccine-hunt/ fluorescence The qPCR 100 0 5 10 15 20 25 30 35 cycle # fluorescence The qPCR 100 10 0 5 10 15 20 25 30 35 cycle # The qPCR fluorescence 1000 100 10 0 5 10 15 20 25 30 35 cycle # fluorescence Key Concept #1: Threshold cycle Ct or Cq : is the PCR cycle at which the amount of PCR product fluorescence equals the threshold. The threshold is the minimal fluorescent value which is significantly greater than background 1000 100 10 threshold 0 5 10 15 20 25 30 35 cycle # Threshold cycle (Ct) Key concept #2: there is a linear relationship between the log of the # of starting molecules and the threshold cycle Log[starting quantity] 10n How to calculate the concentration of an unknown sample using a Standard Curve 1. Plot the standard curve using the values in the Standard Curve table on the excel file (This will be posted on Blackboard). Use the “Scatter plot” with lines connecting the dots from Excel:   2. X axis: Log(Ro) = Log of the starting material Y axis: Ct Average = Ct value corresponding to each concentration of starting material Add a linear trendline. Display the equation and the R-squared value on the chart 3. The linear equation will look like where: 𝑌 = 𝑚𝑋 + 𝑏 𝑌 = 𝐶𝑡 𝑋 = 𝐿𝑜𝑔(𝑅𝑜) Thus, the equation can be re-written as: 𝐶𝑡 = 𝑚 ∗ 𝐿𝑜𝑔 𝑅𝑜 + 𝑏 4. Using this equation, find the concentration of your unknown sample as follows: 𝐶𝑡 − 𝑏 𝐿𝑜𝑔 𝑅𝑜 = 𝑚 Ro = 10[(Ct – b)/b] = the antilog of Log(Ro) Threshold cycle Key concept #3: given the threshold cycle of the PCR reaction you can calculate the starting number of DNA (or RNA) molecules Log[starting quantity] qPCR Instruments 16 Real-time PCR systems consist of THREE main components: 1. Thermal Cycler (PCR machine) 2. Optical Module (to detect fluorescence in the tubes during the run), 3. Computer (to translate the fluorescence data into meaningful results). https://www.google.com/search?q=qpc+instrument&biw=1270&bih=953&source=lnms&tbm=isch&sa=X&ei=0GxfVcKyB7OKsQSWs4GgBA&ved=0CAcQ_AUoAQ#tbm=isch&q=qpcr+result+on+computer Output of Control Standards Melting Curve qPCR – the Concept of MELT CURVES… Melting Curve – raw data RFU Melting Curve – first derivative of the raw data Temperature pUC19 qPCR Protocol Reagents Supplied:      A 48 well plate containing 20 μl/well of 1.25X qPCR Master Mix containing buffer, dNTPs, “Hot Start” Taq Polymerase, Sybr Green, pUC19 Forward Primer, and pUC19 Reverse Primer Serial dilutions of pUC19 have already been added to appropriate wells. Each student will receive an unknown sample of pUC19. Each student will add 5 μl of his/her unknown sample to duplicate wells. The plate will then be placed in the qPCR machine which has been programmed as follows: qPCR Instrument Settings qPCR Instrument Settings 1. 95°C for 10 minutes to activate the polymerase (hot start) – once 2. 95°C for 20 sec – denaturation step 3. 59°C for 30 sec – annealing 40 times 4. 72°C for 30 sec – extension 5. Perform steps 2 through 4 for 40 cycles 6. 72°C for 5 minutes - extension temperature run once 7. Perform melting temperature analysis from 50°C to 94°C in 0.5°C increments, 10 sec at each increment 1 pUC19 2 pUC19 3 Samples 4 Samples 5 Samples 6 Samples A 0.1pg pUC19 0.1 pg pUC19 Student 1 Student 1 Student 8 Student 8 B 1 pg pUC19 1 pg pUC19 Student 2 Student 2 Student 9 Student 9 C 10 pg pUC19 10 pg pUC19 Student 3 Student 3 Student 10 Student 10 D 100 pg pUC19 100 pg pUC19 Student 4 Student 4 Student 11 Student 11 E 1,000 pg pUC19 1,000 pg pUC19 Student 5 Student 5 Student 12 Student12 F 10,000 pg pUC19 10,000 pg pUC19 Student 6 Student 6 Student 13 Student 13 Blank Blank Student 7 Student7 Student 14 Student 14 Blank Blank Blank Blank Blank Blank G H Manipulation of the Data   The data generated by the qPCR instrument will be uploaded as an Excel spreadsheet onto Blackboard Each student will be required to: 1. 2. 3. Generate the standard curve from the data (Cq versus Log10[pg pUC19]) Make a trendline and display the linear equation and the R2 value Use the equation to determine the pg or ng of plasmid DNA in his/her sample Example of Excel Data WELL TARGET A01 A02 B01 B02 C01 C02 D01 D02 E01 E02 F01 F02 Control Control Control Control Control Control Control Control Control Control Control Control WELL TARGET STANDARD CURVE STANDARD CT CT MEAN (Y) NTC 5 pg 50 pg 500 pg 5000 pg 50000 pg N/A N/A 20.09 20.09 17.15 17.22 13.73 13.82 11.31 11.88 7.85 7.77 CT STD. DEV LOG(RO) (X) 0 0 20.09 0 0.69897 17.19 0.047 1.69897 13.78 0.064 2.69897 11.59 0.402 3.69897 7.81 0.06 4.69897 CT SAMPLES SAMPLE CT WELL TARGET SAMPLES SAMPLE A03 Plasmid Student 1 8.18 C05 Plasmid Student 11 13.5 A04 Plasmid Student 2 14.4 C06 Plasmid Student 12 11.5 A05 Plasmid Student 3 11.6 D03 Plasmid Student 13 20.1 A06 Plasmid Student 4 11.7 D04 Plasmid Student 14 17.5 B03 Plasmid Student 5 11.27 D05 Plasmid Student 15 17.4 B04 Plasmid Student 6 14.88 D06 Plasmid Student 16 11.4 B05 Plasmid Student 7 14.21 E03 Plasmid Student 17 20.1 B06 Plasmid Student 8 25.48 E04 Plasmid Student 18 14.0 C03 Plasmid Student 9 18.18 E05 Plasmid Student 19 7.8 C04 Plasmid Student 10 13.5 E06 Plasmid Student 20 11.5 Resources  Packing a chromatography column  https://youtu.be/G4jyd8L0MWE  Performing chromatography  https://youtu.be/VP6Px8zTDNM  Performing SDS-PAGE  https://youtu.be/eaETFKXtNRA  Real Time PCR animation  https://www.youtube.com/watch?v=EaGH1eKfvC0
Standard Curve Concentration Ct Value 10,000pg 9,95 10,000pg 10,19 1000 pg 15,01 1000 pg 15,97 100pg 17,21 100pg 17,32 10pg 20,2 10pg 20,02 1pg 24,67 1pg 24,54 0.1pg 29,35 0.1pg 28,21 Students Student Ct Value Student 1 9,82 Student 1 10,79 Student 2 16,6 Student 2 17,36 Student 3 27,29 Student 3 29,92 Student 4 20,22 Student 4 20,14 Student 5 20,26 Student 5 20,22 Student 6 25,32 Student 6 25,54 Student 7 25,76 Student 7 25,7 Student 8 27,35 Student 8 27,11 Student 9 17,19 Student 9 17,8 Student 10 10,5 Student 10 27,18 Student 11 27,46 Student 11 27,46 Student 12 16,6 Student 12 15,91 Student 13 15,97 Student 13 15,8 Student 14 27,29 Student 14 27,89

Tutor Answer

profwilliams
School: University of Maryland

Attached.

Objectives
The objective of this real time quantitative PCR experiment is to estimate the concentration of
DNA molecules in an unknown sample. The experiment aims to use a qPCR machine and a
standard curve to estimate the concentration of DNA in an unknown sample of Puc19. The qPCR
method measures the amplification of targeted DNA molecules during a polymerase chain
reaction.
Background
The quantitative PCR is an important molecular biology tool commonly used to investigate gene
expression. It measures the quantity of PCR at every cycle of amplification. The products of
amplification are measured as they are being produced using a fluorescent label. A fluorescent
dye, Ethidium Bromide and SyBr Green, bind to dsDNA either directly or indirectly during
amplification through a labeled hydrolyzing probe and emit light when illuminated with specific
wavelength. The fluorescence values are recorded with each cycle of amplification. The
fluorescence signal is directly proportional to the...

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Review

Anonymous
Thanks, good work

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