Bio 140.02 Workflow Module 3 plasmid DNA postlab PDF

Workflow for ~ Module 3: Sample used: recombinant E. coli with pET28a-sfGFP DNA...

Workflow for ~ Module 3: Sample used: recombinant E. coli with pET28a-sfGFP DNA 2 introduced DNA from other species Bacterial culture Overnight culture of recombinant E. coli Extraction of plasmid DNA Amplification of the sfGFP gene insert Plasmid DNA Extraction via Alkali Lysis Polymerase Chain Reaction Visualization of extracted DNA Agarose Gel Electrophoresis Spectrophotometric Analysis of Extracted DNA Ultraviolet Spectrophotometry  www.quora.com/How-is-DNA-different-in-prokaryotes-and-eukaryotes negativecharge enphosphate ↑ polymer = polar [ hitrybaseSi, Anti-paralled a Joisted helix into a Single-stranded DNA: Double-stranded DNA: DNA double helix: Prokaryotes (Bacteria and Archaea) Eukaryotes have a nucleus, and have no nucleus, genomic DNA is genomic DNA is linear and circular and “naked” organized as chromatin "coiled around history proteins Prokaryotes and eukaryotes have non-genomic DNA. J mitoch plasmidy( & chorplant  DNA Extraction Source: Cell cultures: Plant/animal tissue: Forensic evidence: Environmental samples https://labshakers.wordpress.com/ www.youtube.com/watch?v=xlrwef2Y3f0 https://bigdata.cgiar.org/ https://sciencing.com/ https://www.deq.ok.gov/ (pure source I well wall harder to Rondi long yung sample Lentire biome lyse high incentrations a relatively pre ~ won't interfere long ↳ as an it we further methods camptis) OBJECTIVE: high recovery of DNA with no impurities and inhibitors applicable for downstream processes DNA Extraction OBJECTIVE: high recovery of DNA with no impurities and inhibitors applicable for downstream processes General steps: 1. Collection of cells /centrifugation pellet)> - 2. Cell lysis of cells which releases the DNA (and other cellular contents) prote: cell wall a membrane 3. Separation of DNA from other -through reagents /or centrifugated and cellular material 4. Precipitation and concentration of DNA.  DNA Extraction OBJECTIVE: high recovery of DNA with no impurities and inhibitors applicable for downstream processes Recombinant bacterial cell cultures: General steps: 1. Collection of cells 2. Cell lysis of cells which releases the DNA (and other cellular contents) https://labshakers.wordpress.com/ 3. Separation of DNA from other cellular material 4. Precipitation and concentration of DNA.  I markers = ampicillin resistance Plasmid DNA  Plasmid DNA Lextra chromosomal ANA DNA reparated from generic independent replication ~ covalently closed: circular -naturally occuting or synth-via recog. DNA Jech (otoning) Plasmid DNA Plasmid DNA extraction via Alkali Lysis Workflow: OBJECTIVE: recovery of DNA applicable for downstream processes (with no impurities and inhibitors to inhibit PCR) cresuspension) inc - osmotic pressure Al regulator chelator : binds divalent ions (cofactors of nucleased Calkali lysis) & MWANS denatured alkali = makessample ; denatures proteins ; lytis (neutralization soln) dissolved 7 ↓ pl (acidic) AND and precip of > - Yields plasmid complexes (IMWANAd proteins) ] precip plasmid ANA from soon store plasmid here pl ph , chelator Recombinant Escherichia coli https://labshakers.wordpress.com/ Cultured in LB Broth with Kanamycin Plasmid DNA ' Danamyin s orig of replic. https://www.addgene.org/85492 DNA Extraction: next steps Visualization: agarose gel electrophoresis Downstream applications: Amplification via PCR DNA sequencing Cloning for recombinant DNA applications Quantitative/Qualitative analysis: UV spectrophotometry  Workflow for Module 3: Sample used: recombinant E. coli with pET28a-sfGFP DNA Bacterial culture Overnight culture of recombinant E. coli Extraction of plasmid DNA Amplification of the sfGFP gene insert Plasmid DNA Extraction via Alkali Lysis Polymerase Chain Reaction Visualization of extracted DNA Agarose Gel Electrophoresis Spectrophotometric Analysis of Extracted DNA Ultraviolet Spectrophotometry Electrophoresis * 0008-yellow · : 88-bhe = -wood - small travels Electrophoresis is the migration through a matrix of charged faster d molecules in solution by applying an electric field farther Speed of migration: depends on charge, shape, size of the molecule; and the gel concentration Molecular separation technique Agarose gel: Source: https://www.wisegeek.com/what-is-agarose-gel.htm Agarose is a linear polysaccharide extracted from red marine algae D-galactose 3,6-anhydro-L-galactose DNA and RNA: migration thru the gel -much ↑ pares - ↓ van ↓ powers ↑ conot usgen ↳ bacir (bp) , small 1 kb Plus DNA Marker, NEB www.neb.com https://thebumblingbiochemist.com/ https://bit.ly/2Oujks8  Agarose gel electrophoresis (AGE) OBJECTIVE: confirm the success of genomic DNA extraction  Agarose gel preparation  Sample loading and electrophoresis  Visualization https://commons.wikimedia.org/wiki/File:Loading_DNA_sample _into_to_agarose_gel_electrophoresis.jpg Electrophoresis components Source: http://www.mupid.com/ Source: UV excites LEBr for visualization  but Buffer system: Loading buffers: > - not stain lotoes only for visualization of movement Tris-acetate-EDTA (TAE) Blue Tris-borate-EDTA (TBE) Tracking dyes Glycerol ine density - Buffer/ddH2O para di magfloat Source: Agarose Gel Electrophoresis Tip #1: TAE vs TBE https://youtu.be/eQqhOSp80Qg Source: https://commons.wikimedia.org/wiki/File:Loading_DNA_sample_i nto_to_agarose_gel_electrophoresis.jpg Visualizing DNA: WARNING: Ethidium bromide is highly MUTAGENIC!  Gel Red  ↑makakapa Gel Red  molecules it Safer alternative to ethidium bromide: Gel Red  L nuc-acids IUPAC name: 5,5'-(6,22-dioxo-11,14,17-trioxa-7,21- diazaheptacosane-1,27-diyl)bis(3,8-diamino-6- phenylphenanthridin-5-ium) iodide Other names: Dye No. 35  louent crosslinking permanent get NUC - form Dada-larger SDS-PAGE vs AGE, Protein vs NA proteins - maller I will meld is Resolving Power neated Molecular Weight vertical horizontal http://book.bionumbers.org/ https://thebumblingbiochemist.com/  DNA supercoiling: Circular DNA ladder MW' = linear Plasmid-cir  Open circular (relaxed) of is d I is MW need kaioil just for ng tranking of micle weight Movement & same plazaid  Supercoiled of is M sila  Plasmid DNA (pET28a-sfGFP, MW 5975bp ) was extracted from recombinant pET28a-sfGFP, MW 5975bp E. coli via Alkali Lysis, run in a 1% agarose gel at 100 v for 45 minutes, and stained with ethidium bromide. For all samples, 5 ul was mixed with 2 ul gel loading dye and the entire volume loaded into the gel. The MW marker used was CSL-MDNA-1K (Cleaver Scientific) Group 1 Group 2 1 2 3 4 5 1 2 3 4 5 a Hand camd MW marker guide CSL-MDNA-1K A s degraded near I Bio 140.02 SU, 1st Sem, SY 24-25  Plasmid DNA (pET28a-sfGFP, MW 5975bp ) was extracted from recombinant pET28a-sfGFP, MW 5975bp E. coli via Alkali Lysis, run in a 1% agarose gel at 100 v for 45 minutes, and stained with ethidium bromide. For all samples, 5 ul was mixed with 2 ul gel loading dye and the entire volume loaded into the gel. The MW marker used was CSL-MDNA-1K (Cleaver Scientific) Group 3 Group 4 1 2 3 4 5 1 2 3 4 5 MW marker guide CSL-MDNA-1K Bio 140.02 SU, 1st Sem, SY 24-25  Workflow for Module 3: Sample used: recombinant E. coli with pET28a-sfGFP DNA Bacterial culture Overnight culture of recombinant E. coli Extraction of plasmid DNA Amplification of the sfGFP gene insert Plasmid DNA Extraction via Alkali Lysis Polymerase Chain Reaction Visualization of extracted DNA Agarose Gel Electrophoresis Spectrophotometric Analysis of Extracted DNA Ultraviolet Spectrophotometry The electromagnetic spectrum Many biological molecules intrinsically absorb light in the UV range Absorption can be measured with a spectrophotometer https://imagine.gsfc.nasa.gov/science/toolbox/emspectrum1.html Basic structure of spectrophotometers (illustrated by Heesung Shim, https://chem.libretexts.org/ ) UV Spectrophotometry A UV-VIS spectrophotometer and cuvettes www.medicalexpo.com 100 µL – 1000 µL sample volume NabiTM UV VIS nano Basic structure of spectrophotometers spectrophotometer (illustrated by Heesung Shim, https://chem.libretexts.org/ ) www.md-best.com/nabi.html/ 0.5 µL – 2.0 µL sample volume Microvolume spectrophotometers have allowed the reading of absorbance of samples without the need for containment devices like cuvettes or capillaries DNA UV Spectrophotometry Workflow: OBJECTIVE: check purity and concentration of DNA extract NabiTM UV VIS nano spectrophotometer www.md-best.com/nabi.html/ 0.5 µL – 2.0 µL sample volume  DNA and UV Spectophotometry Nitrogenous bases Protein backbone, salts, phenol Amino Acids (aromatic rings) 260 nm 230 nm 280 nm because dis nitro peal maxabs & zoonuh o lit because o - - - max abs & 280 Depfre as I because of amae ↳ www.biotek.com/assets/tech_resources/A260A280_Spectral_Scanning_App_Note.pdf  UV Spectrophotometry to assess purity Pure preparation of DNA: 1.8 < (Abs260/Abs280) < 1.9 Protein contamination: (Abs260/Abs280) < 1.8 RNA: (Abs260/Abs280) > 2.0 Pure preparation of DNA: 2.3 < (Abs260/Abs230) < 2.4 ⑧ l used secondary as measure … and to estimate concentration [dsDNA] = (Abs260) x (dilution factor) x (50 µg/mL) Where: dilution factor = total volume of diluted solution/volume of sample UV Spectrophotometry to estimate concentration [dsDNA] = (Abs260) x (dilution factor) x (50 µg/mL) Where: dilution factor = total volume of diluted solution/volume of sample Derived from the Beer-Lambert law, the amount of light absorbed at 260 nm is proportional to the concentration of nucleic acid in solution. Extinction coefficients have been determined for dsDNA, RNA, and ssDNA using a 10 mm path length and allow the creation of conversion factors in the absence of a standard curve. For example, a dsDNA sample with A260 = 1 will have a concentration of 50 ng/µl (for pure samples only!) DNA UV Spectrophotometry Workflow: OBJECTIVE: check purity and concentration of DNA extract it is blank needfera no NabiTM UV VIS nano spectrophotometer www.md-best.com/nabi.html/ 0.5 µL – 2.0 µL sample volume  Class Results Sample Abs 230 Abs 260 Abs 280 Abs260/ Abs 260/ Purity Estimated Abs280 Abs230 ng/uL DNA  Workflow for Module 3: Sample used: recombinant E. coli with pET28a-sfGFP DNA Bacterial culture Overnight culture of recombinant E. coli Extraction of plasmid DNA Amplification of the sfGFP gene insert Plasmid DNA Extraction via Alkali Lysis Polymerase Chain Reaction Visualization of extracted DNA Agarose Gel Electrophoresis Spectrophotometric Analysis of Extracted DNA Ultraviolet Spectrophotometry  All notes are on Polymerase Chain Reaction (PCR) cellmo) notebook In vitro cloning of DNA using Kary Mullis (1944 – 2019) Invented PCR in 1983 a thermostable DNA Nobel Prize (Chemistry) 1993 polymerase C:\Documents and Settings\AMOR\My Documents\RAs folder\trinket\MBB Primer (VDM)\ver.2\PCR.jpg  Polymerase Chain Reaction (PCR) Thermal cycling: PCR uses repeated rounds of: Denaturation 1. Denaturation, of dsDNA Extension of primers by DNA pol 2. Annealing, and (“elongation”) 3. Elongation Hybridization to amplify DNA (“annealing”) of primers  Polymerase Chain Reaction (PCR) Each cycle doubles the amount of DNA in the previous cycle to that after a few cycles, the predominant DNA in the reaction is identical to the sequence specified by the primer pair.  Sample Results: PCR of plasmid DNA DNA Template DNA used PCR amplification product Dilute DNA to use as template PCR: amplify gene insert 23,130 bp 21,226 bp 2,322 564 1,584 900 bp Target for amplification using forward and reverse primers is indicated in RED, expected MW of PCR product is approximately 900 bp.  Optimization of PCR reaction mixes and cycling parameters: PCR experiments often have to be OPTIMIZED so that stringent conditions yield specific products. Components of PCR Source: www.goldbio.com/goldbios-pcr-overview  PCR experiments often have to be Trouble shooting PCR OPTIMIZED so that stringent conditions yield specific products. Common problems Things to check: No product/low DNA template amplification Primers Incorrect product size Other reaction Multiple bands/non- components: specific amplification/ Mg2+ smears Polymerase Primer dimers Contaminants in the reaction False positive (negative control amplifies) Cycling parameters Technical References: https://international.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular- biology/pcr-education/pcr-reagents-enzymes/pcr-troubleshooting.html Polymerase Chain Reaction (PCR) OBJECTIVE: amplify the uperfolder GFP gene insert in the pET28 plasmid Plasmid DNA - PCR Template DNA Target for amplification using T7 forward and reverse primers is indicated in RED, expected MW of PCR product is approximately 900 bp. https://www.addgene.org/85492 Polymerase Chain Reaction (PCR) PCR cycling parameters: Initial denaturation 94C 30 secn 27 cycles: Denaturation 94C 30 sec Annealing 41C 40 sec Extension 68C 60 sec Final extension 68C 5 min Hold 4C  ֹֹ Horn, I. R., Verleg, P. A., Ibrahim, N. Z., Soeleman, K., van Kampen, F., Ruesen, M. O.,... & Gravendeel, B. (2020). Mushroom DNA barcoding project: Sequencing a segment of the 28S rRNA gene. Biochemistry and Molecular Biology Education, 48(4), 404- 410. https://doi.org/10.1002/bmb.21388  Molecular Weight Marker Guide, VC 1kb DNA Class result: Groups 4 and 8 pET28a-sfGFP, MW 5975bp Target for amplification using T7 forward and reverse primers is indicated in RED, expected MW of PCR product is approximately 900 bp.  Virtual Lab: Bacterial ID https://www.biointeractive.org/classroom-resources/bacterial-identification-virtual-lab Overall objective of virtual lab ? https://en.wikipedia.org/wiki/DNA_barcoding  PCR & DNA Barcoding https://doi.org/10.3389/fevo.2020.00028 https://blog.nationalgeographic.org/2017/06/16/sushi-roulette- is-the-fish-you-ordered-the-one-you-got/ https://www.boredpanda.com/fish-fraud-dna-test-biology-students-homework/ https://twitter.com/AwesomeBioTA https://www.eurekalert.org/news-releases/938896 Barcoding of Life: digital identification for life http://www.ibol.org/  The chemistry of DNA synthesis DEOXYNUCLEOSIDE TRIPHOSPHATES (dNTPs) DIDEOXYNUCLEOSIDE TRIPHOSPHATES (ddNTPs) Sanger sequencing (aka chain- termination or dideoxy H sequencing) which makes use of ddNTPs. Chain-termination sequencing (automated) 1. Reaction components Template DNA (to be sequenced) DNA polymerase Oligonucleotide primer Standard dNTPs ddNTPs – each labelled with a different fluorescent marker Thermal cycle sequencing 2. Separation of fragments using capillary gel electrophoresis Source: Automated DNA Sequencing www.magazinescience.com/en/biology/automated-dna-sequencing/  www.wired.com/2008/07/british-institu/ Source: Automated DNA Sequencing www.magazinescience.com/en/biology/automated-dna-sequencing/ Sequencing files (results) 1. Chromatogram (.abi,.pdf) 2. DNA sequence (FASTA,.txt) Sample files from ZAN Maini NGS: next generation sequencing (current, 2 nd gen) High throughput Micro- and nano-technology Different technologies by different companies Sequencing of DNA libraries “shotgun,” “parallel” Massive parallel sequencing = A LOT OF DATA Source: “The third wave of next generation sequencing” https://bit.ly/3rkNf3I Reduce sample size, cost and Third gen: time Single molecule sequencing Information databases: public domain The International Nucleotide Sequence Database Collaboration is the core infrastructure for sharing nucleotide sequence data in the public domain The total size of sequence data maintained by the INSDC has exceeded 9 petabytes in 2020. BLAST  BLAST “find regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.” BLAST https://microbenotes.com/bioinformatics-introduction-and-applications/https://microbenotes.com/bioinformatics-introduction-and-applications/