Term 1 Lab 3 ELN PDF

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electronic lab notebook for lab 3...

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Term 1 Lab 3 ELN In preparation for the lab day, please read the lab 1 entry in the Biochemistry 2L06 courseware (posted in the “course information” folder of A2L) and fill out the following sections. Thank you. This ELN is due Wednesday October 28, no later than 11:00 am in the appropriate assignment folder on A2L. This is an individual assignment. Please submit one pdf file of this completed template.

Purpose and experimental overview In the space provided below: 1. Purpose - Briefly describe the purpose of each part of this lab. What is your overall goal for this lab day (very concise, cannot exceed 250 words)? There are 3 parts to this lab- purification of the digested amplicon, DNA ligation, and bacterial transformation. After the previous lab where we performed PCR and cut out agarose bands from the gel, we must separate the DNA and the agarose gel in order to extract the DNA fragment. This is done using a DNA gel extraction kit such as the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit, which was used in our lab. After we have extracted the DNA fragment, we are able to ligate the backbone DNA with our folA  insert DNA. The ligation process (incorporating the folA g  ene into the pET26b plasmid) is performed by the T4 DNA ligase. The reaction will occur in five separate tubes (inhouse primers, team-designed primers and control reaction tubes), which contain the backbone DNA, folA i nsert DNA, 5X ligase buffer, T4 DNA ligase, and water. After ligation has been completed, we will perform bacterial transformation into E. coli  DH5α chemically-competent cells in order for bacterial colonies to grow exponentially. Firstly, 5 aliquots (40 μL) of E. coli  DH5α chemically-competent cells are thawed on ice. Each ligation reaction (2.5 μL) is added to one aliquot and thawed on ice for 10 minutes, heat shocked at 42°C for 42 seconds, then placed back onto ice. Liquid LB media is added to the microcentrifuge tubes, allowing the bacteria to absorb nutrients after experiencing heat shock. Lastly, the supernatant from the centrifuged tubes are placed on separate LB agar plates so the E.coli containing the folA g  ene can multiply.

2. In your own words, please describe how the “DNA extraction from agarose gel” procedure works (max 500 words, you can also use a flowchart to describe this if you wish). To help you with this task, please read the following resources: ·

PureLink™ Quick Gel Extraction and PCR Purification Combo Kit manual found here: http://products.invitrogen.com/ivgn/product/K220001 ) - please note that you will be using the gel extraction protocol not the PCR purification protocol.

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https://bitesizebio.com/13533/how-dna-gel-extraction-works/ Firstly, the DNA bands must be excised from the agarose gel electrophoresis apparatus  using a scalpel. In order to extract the DNA from agarose gel, the gel must first be dissolved so that the fragments of DNA can be separated from the gel molecules using centrifugation. By discarding the flow through in several steps during the centrifugation stage, other elements are discarded from the sample and purification of the DNA is accomplished. After weighing the gel piece and the microcentrifuge tube both separately and together, L3 buffer (Gel Solubilization Buffer) is placed into the microcentrifuge tube. This allows the gel slice to dissolve. The tube is placed into a heat block at 50°C for 10 minutes to incubate. When the contents of the microcentrifuge tube appear to be dissolved, it is placed into the heat block for five minutes to incubate further. 1 gel volume of isopropanol is added to the sample (1:1 ratio of isopropanol to gel slice). The tube is then placed in a centrifuge at >10,000 × g for one minute, and the flow through is discarded. Then, a mixture of ethanol and the wash buffer W1 from the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit is added and the sample is centrifuged again for one minute. After the flow through is discarded, the sample is centrifuged for 2-3 minutes to separate the DNA fragments from the buffer and ethanol. Afterwards, 50 μL Elution Buffer (E1) is added to the sample and incubated for one minute at room temperature, then centrifuged for one minute which produces the purified DNA.

Marking Scheme (for lab mentor use only) – for the purpose and experimental overview section, please provide a mark out of 4, whereby: Purpose - Is concise, uses proper grammar/scientific terminology, and appropriately describes the day’s lab.

0-0.5 0.5-1 1-1.5 1.5-2 Unacceptable Sufficient Accomplished Exemplary *

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Mark (/2) and comments: “DNA extraction from agarose gel” explanation – Is easy to understand, clearly explains the main point of the technique.

0-0.5 0.5-1 1-1.5 1.5-2 Unacceptable Sufficient Accomplished Exemplary *

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Calculations and background knowledge Please ensure that you have completed all required calculations in your courseware PRIOR to the lab time. Your lab mentors will check your courseware calculations at the start of the lab. This is part of your participation mark. Additionally, please complete the following as part of your ELN requirements:

Background information: In this course you will utilize a spectrophotometer to measure a myriad of things: DNA concentrations, protein concentrations, cells, etc. Spectrophotometers come in many shapes and sizes, but they are basically an analytical instrument used to measure and estimate the concentration of an analyte in solution. “Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation) over a certain range of wavelength. Spectrophotometry is a measurement of how much a chemical substance absorbs or transmits. A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected. For each wavelength of light passing through the spectrometer, the intensity of the light passing through the reference cell is measured. “(Excerpt obtained directly from The LibreTexts libraries (spectrophotometry and the Beer-Lambert law chapters). LibreTexts content is licensed by C  C BY-NC-SA 3.0 . ) Image created by Felicia. Red arrow provided by Presenter Media, © 2009-2020 Eclipse Digital Imaging, Inc.

In this diagram Io = the initial, incident light and I = the final transmitted light once the sample absorbed some of the intensity, as is the case for the example depicted here. Therefore, if I < Io, we can infer that the sample absorbed some of the light. This measurement is then converted to an absorbance by the spectrophotometer as defined by the incident intensity (Io ) and transmitted intensity (I). The equation is as follows: If we have the exact same value for both intensities (meaning that our sample did not absorb), we obtain an absorbance of zero as log10 (1) = 0. In this course we will not be using this equation directly as our spectrophotometers will convert this for us, and we only deal with the final absorbance values.

For measuring nucleic acid concentrations we often times use a microspectrophotometer, also known as a nanodrop: Question 1: You have just purified your double-stranded plasmid DNA and measured the absorbance readings on the nanodrop. You obtained the following values from the 260 and 280nm wavelengths: A260 = 3.9 A280 = 2.1 What is the concentration of DNA in the original, undiluted, double stranded DNA sample (express this in ng/µL)? Please show your math/reasoning. (2 marks)

Concentration of DNA = Value @A

X 50 µg/ml

260nm 

A260 = 3.9 x 50 µg/ml = 195 ng/µL

Question 1b: Is this double stranded (ds) DNA sample pure? Please explain and show your math/reasoning (1 mark) A280 = 2.1 x 50 µg/ml = 105 ng/µL A260/A280 = 1.857 This double stranded DNA sample is pure because a relatively pure DNA sample has a A260/A280 ratio of approximately 1.8. This is to find whether there are many contaminants in the sample or not- higher ratios suggest that more RNA is present in the DNA sample, while lower ratios suggest the presence of more protein. Question 2: When performing ligations, different insert: plasmid ratios are tested. These ratios are calculated as such: Let’s say you want a 1:1 ratio of insert to plasmid and you have the following information. Please calculate how much insert (in ng) you need for a 1:1 ligation reaction (please show your math). (2 marks) Digested pET26 plasmid = ~ 5000 bp (plasmid size) Digested folA insert = ~ 500 bp (insert size) Concentration of pET26b plasmid = 20 ng/µL. You will be using a total of 4 µL in your reaction. Total concentration of pET26b plasmid = 20 ng/uL x 4 uL = 80 ng concentration of insert (ng ) concentration of plasmid (ng ) = insert size (bp) plasmid size (bp) x 500 bp

x=8

=

80 ng 5000 bp

(for a 1:1 ratio of insert to plasmid)

Question 3: In your own words, please briefly (3-4 sentences or point form) describe the following plasmid features and why they are important for your system (i.e. cloning and expressing your gene of interest). Additionally, please include the pET26b (Novagen) base pair (bp) location for each feature (range, where applicable. The pBR322 ori has a start bp only). Hint: please use the posted pET26b plasmid map posted in the A2L week 7 ELN-3 folder to help you answer some of these questions. For the rest you can conduct Google searches. Please make sure you reference appropriately. (3 marks, 0.5 mark/feature)

a. pBR322 origin of replication (i.e. replicon) - 3277 bp The origin of replication is where the DNA begins replication and synthesis of a new strand is started. It is a specific sequence that allow replication initiator factors to attach and signal DNA replication. This sequence is important for cloning and expressing the folA  gene because after the folA g  ene is ligated to the plasmid, subsequent cloning and production of more plasmids will begin at the ori site and allow the production of many more folA  genes. b. kanR  gene – 3986-4798 bp

The kanR g  ene is the resistance gene present in the pET26b plasmid and it provides resistance for the plasmid against the kanamycin antibiotic by producing a protein product that inactivates kanamycin. This will be present in the growth media of our sample and it is important to have this resistance gene as a way of selecting against bacteria that do not have the plasmid. Without the plasmid, they will not have the resistance gene and will die when placed in kanamycin-supplemented growth media. This is important for cloning the folA g  ene because we must remove unsuccessful colonies of bacteria (do not contain the folA g  ene and will not produce clones). c. multiple cloning site (MCS) – 158-225 bp This is a sequence of DNA in the plasmid that contains multiple restriction enzyme cut sites. In the pET26b plasmid, there are 10 unique cut sites for different restriction endonucleases and this allows for cutting of the plasmid using a specific restriction endonuclease. This is important for cloning and expression of the folA gene because we must cleave the folA  gene and the pET26b plasmid using the same restriction enzymes so that they have the same cut sites with complementary sticky ends, allowing the ligation of the folA gene into the digested pET26b plasmid. Only after this step are we able to clone the gene of interest, since it has been ligated into the plasmid. d. His tag – 140-157 bp His tag is a DNA sequence that has six to nine histidine residues and is used for IMAC purification (based on affinity to metal ions) and identification of DNA molecules. After the insertion of the folA g  ene in the pET26b plasmid, a recombinant C-terminal His-tagged protein will be expressed. This is important in cloning because we are able to separate the bacteria using the His tag and purification of the sample can be done, separating the plasmid DNA from other components in the sample that may be contaminants such as other proteins. e. T7 promoter – 361-377 bp The T7 promoter is a sequence that is recognized by the T7 RNA polymerase, which attaches to the T7 promoter on the plasmid and begins transcription. This is important in cloning because the DNA containing the folA  gene must be first transcribed, then translated, in order for a new daughter strand of DNA to be produced. The T7 promoter allows for transcription of the parent DNA strand, which creates many daughter strands which all contain the gene of interest. f.

pelB leader sequence – 224-289 bp

The pelB leader sequence is a specific DNA sequence that directs a polypeptide to the bacterial periplasm, allowing it to fold more efficiently. This sequence is not used for our purposes in the 2L06 labs and cloning.

References Please provide your reference list below. Please make sure you embed in-text citations throughout the ELN and you use the APA citation style. His-tagged Proteins–Production and Purification: Thermo Fisher Scientific - US. (n.d.). Retrieved October 28, 2020, from https://www.thermofisher.com/hk/en/home/life-science/protein-biology/protein-biology-learning-center/pr otein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html

PureLink® Quick Gel Extraction and PCR Purification Combo Kit. (2011, June 13). Retrieved October 28, 2020, from https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets. thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fpurelink_gel_extraction_pcr_combo_man.p df Vulcu, F. (2020). Biochemistry 2L06 courseware. Retrieved October 28, 2020, from https://avenue.cllmcmaster.ca/d2l/le/content/334928/viewContent/2755054/View

Overall ELN mark (for lab mentor use only) Overall – This ELN is overall very well 0-0.5 0.5-1 1-1.5 1.5-2 structured and referenced appropriately. Unacceptable Sufficient Accomplished Exemplary *

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ELN TOTAL MARK (for lab mentor use, 14 maximum marks) –

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