Document Details

AmpleDwarf

Uploaded by AmpleDwarf

Loyola Marymount University

Tags

cell biology lab techniques nucleic acids molecular biology

Summary

This document details various lab techniques. It covers cell biology, including methods like hemocytometry and flow cytometry, as well as nucleic acid and protein techniques such as karyotyping and sequencing. The included techniques are critical for molecular biology and genetic research.

Full Transcript

8/27/24, 8:28 PM Platform | Study Fetch Cell Biology Lab Techniques (00:42 - 01:06) Hemocytometers: Grids used to count samples under a microscope Allows you to divide your view into fields and count the number of whatever...

8/27/24, 8:28 PM Platform | Study Fetch Cell Biology Lab Techniques (00:42 - 01:06) Hemocytometers: Grids used to count samples under a microscope Allows you to divide your view into fields and count the number of whatever you're trying to measure Flow Cytometry: More advanced technique similar to a hemocytometer Feeds the sample through a laser light source Funnels individual cells so they can be hit with a laser Allows you to infer different properties of the cells, such as: Granularity Density Size Specific markers Cell Fractionation: Using a centrifuge to spin down a suspension and form a precipitate Different intensities of spinning separate out components of different densities Differential centrifugation: Start with lysed cells with all organelles mixed together Do low-speed centrifugation to pellet whole cells, nuclei, and cytoskeletons Take the supernatant and spin faster to pellet mitochondria, lysosomes, etc. Repeat this process to separate out smaller and smaller cellular components Nucleic Acid and Protein Lab Techniques (03:14 - 05:55) Karyotyping: Laying out all the chromosomes of a species and analyzing chromosome number and structure Sanger Sequencing: Reaction mixture contains normal nucleotides and chain-terminating dideoxynucleotides Synthesis stops when a dideoxynucleotide is incorporated, creating DNA fragments of varying lengths Run these fragments on a gel to determine the sequence by the length of each terminated fragment Limitations of Sanger Sequencing: Resource-intensive to sequence each nucleotide Limited in the length of sequence that can be read Next-Generation Sequencing: Instead of terminating synthesis, a camera detects the color of the fluorescent tag added to each new nucleotide Can continuously monitor DNA synthesis and record the sequence in real-time Allows for much longer sequences to be read https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 1/8 8/27/24, 8:28 PM Platform | Study Fetch Genomics and Miscellaneous Techniques (00:28 - 00:42) Techniques for studying genomics and other miscellaneous lab procedures will be covered in the next part of the lesson. Chemically Protecting DNA Bases and Fluorescent Labeling (00:06:45 - 00:07:01) Chemically protect DNA bases by adding a base, taking a picture, detecting it, adding another base, taking another picture, and repeating the process This allows each new base added to be fluorescently labeled with a different color, creating a "color code" of the DNA sequence Recombinant DNA and Restriction Fragment Length Polymorphisms (00:07:01 - 00:07:22) Recombinant DNA is DNA that has had a new sequence added to it Fluorescent labeling of added bases allows us to determine the sequence of the recombinant DNA (00:07:22 - 00:07:42) Restriction fragment length polymorphisms (RFLPs) use knowledge of restriction sites within the genome to detect differences in DNA sequences A plasmid can be cut using a restriction enzyme, and a new sequence can be inserted into the plasmid (00:07:42 - 00:07:56) Restriction enzymes (nucleases) cut DNA at specific sequences The number of DNA fragments generated can indicate the presence or absence of a specific sequence (00:07:56 - 00:08:08) If a specific sequence is present, the restriction enzyme will cut the DNA, leading to multiple bands If the sequence is absent, the DNA will not be cut, leading to a single band (00:08:08 - 00:08:20) The presence or absence of a specific sequence can be used to determine genotypes or polymorphisms (00:08:20 - 00:08:33) If a mutation destroys a restriction site, the DNA will not be cut, leading to a single band instead of two (00:08:33 - 00:08:46) The loss of a restriction site can be used to identify the presence of a disease-causing mutation (00:08:46 - 00:08:58) If a person has the disease, the DNA will not be cut by the restriction enzyme, leading to a single band https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 2/8 8/27/24, 8:28 PM Platform | Study Fetch If the person is normal, the DNA will be cut, leading to two bands (00:08:58 - 00:09:12) This is a rapid and easy way to identify the loss of a specific genomic region, which may be associated with a disease (00:09:12 - 00:09:25) Southern blotting is a technique used to detect specific DNA sequences in a sample DNA Fingerprinting (00:09:25 - 00:09:41) DNA fingerprinting uses a set of restriction enzymes to digest DNA and detect different polymorphisms (00:09:41 - 00:09:56) Each person has unique restriction sites in their genome, leading to a unique pattern of DNA fragments when digested (00:09:56 - 00:10:09) By comparing the restriction fragment patterns, you can determine if two DNA samples are from the same person (00:10:09 - 00:10:22) DNA fingerprinting can be used for identification, paternity testing, and other forensic applications Polymerase Chain Reaction (PCR) (00:10:22 - 00:10:34) PCR is a technique used to rapidly amplify a specific DNA sequence (00:10:34 - 00:10:46) PCR starts with a DNA template, which is then heated to separate the two strands Primers are designed to bind to specific sequences on the DNA template (00:10:46 - 00:10:58) Primers can be designed to target a specific gene or sequence, or they can be random hexamers that bind throughout the DNA (00:10:58 - 00:11:15) The primers anneal to the DNA template, and new DNA strands are synthesized The cycle of heating, annealing, and synthesis is repeated to exponentially amplify the target sequence (00:11:15 - 00:11:30) The newly synthesized DNA strands are then used as templates for the next round of amplification (00:11:30 - 00:11:42) This rapid amplification allows for the production of many copies of a specific DNA sequence Molecular Cloning https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 3/8 8/27/24, 8:28 PM Platform | Study Fetch (00:11:42 - 00:11:56) Molecular cloning is a technique used to produce multiple copies of a gene or DNA sequence PCR can be used to amplify a specific gene, which can then be used for further experiments (00:11:56 - 00:12:22) A plasmid vector is used to clone the amplified gene The plasmid contains an antibiotic resistance gene and a gene for blue/white screening (e.g., lacZ) (00:12:22 - 00:12:32) The amplified gene of interest (e.g., Hoxa1) can be inserted into the plasmid vector (00:12:32 - 00:12:57) The plasmid with the inserted gene can then be used to transform bacteria or other cells, allowing for the overexpression of the gene of interest Plasmids and Genetic Engineering Inserting a Gene of Interest into a Plasmid (00:13:13 - 00:13:31) We have a plasmid with a lac Z gene that contains a restriction site We will insert our construct (gene of interest) into this restriction site within the lac Z gene (00:13:31 - 00:13:50) We will cut the lac Z site using a restriction enzyme We can then insert the sequence we want to test into this site This allows us to create a new plasmid with our gene of interest (00:13:50 - 00:14:07) We can then take this new plasmid and introduce it into an experimental model, such as cell culture or bacteria We can add an antibiotic to the culture, which will kill off any bacteria that don't have our plasmid (00:14:07 - 00:14:34) However, there is a problem - some plasmids may have just the backbone without our gene of interest, but still have the antibiotic resistance These plasmids would survive the antibiotic selection (00:14:34 - 00:14:57) The lac Z gene is used to screen out these unwanted plasmids In cells/bacteria with our gene of interest, the lac Z https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 4/8 8/27/24, 8:28 PM Platform | Study Fetch gene will be disrupted and won't function Cells with the unmodified plasmid will have a functional lac Z gene and turn blue (00:14:57 - 00:15:25) We can then select for the white colonies, which indicates the plasmid has our gene of interest inserted These colonies will be antibiotic resistant (from the plasmid) but not have a functional lac Z gene (00:15:25 - 00:15:40) This allows us to isolate and grow out large quantities of the plasmid with our gene of interest We can then use this plasmid for further experiments (00:15:40 - 00:16:00) Plasmids with an uninterrupted lac Z gene will turn blue Plasmids with the inserted gene will have a disrupted lac Z and remain white Bacteria without any plasmid will also remain their natural color and die from the antibiotics (00:16:00 - 00:16:11) Understanding plasmids as vectors and how to integrate genes of interest is an important concept Gel Electrophoresis (00:16:11 - 00:16:26) DNA is negatively charged, allowing it to be separated by running it through an electric current Gel electrophoresis is an important technique to understand (00:16:26 - 00:16:56) DNA is loaded into an agarose gel, which acts as a sieve When an electric current is applied, the DNA will migrate through the gel Larger DNA fragments will move more slowly through the gel due to greater friction and resistance (00:16:56 - 00:17:24) This creates a gradient of DNA fragment sizes, with smaller fragments moving further down the gel The distance traveled by each fragment can be used to determine its approximate size Southern Blotting (00:17:24 - 00:17:48) Southern blotting is a technique to detect specific DNA sequences After running DNA on a gel, the fragments are transferred to a nitrocellulose membrane The membrane is then probed with labeled, complementary DNA sequences (00:17:48 - 00:18:20) The probes will hybridize to their complementary sequences on the membrane https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 5/8 8/27/24, 8:28 PM Platform | Study Fetch This allows the detection and identification of specific DNA fragments based on their size and the probe used (00:18:20 - 00:18:52) The size of the DNA fragments can be determined by comparing to a DNA ladder or size standard This provides information about the length and presence of specific DNA sequences Northern Blotting (00:18:52 - 00:19:16) Northern blotting is similar to Southern blotting, but uses RNA instead of DNA RNA is extracted and run on a gel, then transferred to a membrane and probed (00:19:16 - 00:19:46) RNA is more unstable and reactive than DNA, so special care must be taken to prevent degradation RNA-degrading enzymes are ubiquitous in the environment and can quickly break down RNA samples Transcript Notes: Blotting Techniques RNA and Protein Integrity (00:19:58 - 00:20:13) RNA is very easily degraded, so you must be very careful when working with RNA samples Loss of sample quality and integrity is a major concern when doing northern blotting or any RNA-based work Steps in Southern and Northern Blotting (00:20:13 - 00:20:27) Mnemonic: "Come get the right visuals" Steps: 1. Cleave the DNA (for Southern blot) 2. Gel electrophoresis 3. Transfer the fragments onto a membrane (typically nitrocellulose) 4. Radioactively or fluorescently label the DNA probe 5. Visualize the DNA probe using X-rays or a fluorescent camera Western Blotting (00:20:27 - 00:21:32) Used to identify proteins, not nucleic acids Steps: 1. Prepare the protein sample, often by denaturing it 2. Separate the proteins by size using SDS-PAGE 3. Transfer the separated proteins to a membrane (typically nitrocellulose) 4. Incubate the membrane with antibodies specific to the proteins of interest 5. Detect the antibody-bound proteins, often using chemiluminescence or fluorescence https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 6/8 8/27/24, 8:28 PM Platform | Study Fetch Antibody Detection in Western Blotting (00:21:32 - 00:22:56) Antibodies are used to detect the proteins on the membrane Primary antibodies bind directly to the proteins Secondary antibodies bind to the primary antibodies and are labeled for detection Labeling can be with radioactivity, fluorescence, or enzyme-linked substrates that produce light Quantification of Western Blots (00:22:56 - 00:23:10) The detected bands can be quantified to determine the relative amounts of each protein in the sample Mnemonic for Blotting Techniques (00:23:10 - 00:23:52) Southern Blot: DNA, Separation, Detection Northern Blot: RNA, Separation, Detection Western Blot: Protein, Separation, Detection ELISA: Enzyme-Linked Immunosorbent Assay ELISA vs Western Blot (00:23:52 - 00:25:19) ELISA is similar to Western blot but uses immobilized antibodies instead of gel separation Proteins bind to the immobilized antibodies, then a labeled secondary antibody is added for detection ELISA is higher-throughput compared to Western blot ELISA Formats (00:25:40 - 00:26:18) Direct ELISA: Immobilized antigen, labeled secondary antibody Indirect ELISA: Immobilized antigen, primary then labeled secondary antibody Indirect allows for signal amplification compared to direct DNA Microarrays (00:26:29 - 00:26:54) DNA sequences are immobilized on a chip Used to measure expression levels of many genes in a sample DNA Microarray Analysis (00:26:54 - 00:27:09) Break up your DNA Hybridize it to a DNA chip Detect any known sequences that have hybridized to the chip Challenges of DNA Microarrays(00:27:09 - 00:27:20) DNA microarray analysis is expensive Other sequencing and detection techniques are more commonly used now https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 7/8 8/27/24, 8:28 PM Platform | Study Fetch DNA Microarray Data(00:27:20 - 00:27:33) DNA microarrays can also be used for RNA analysis Produces an intensity graph for each probe on the array "Crazy heat plot" shows intensity of each known sequence in the sample Interpreting Microarray Data(00:27:33 - 00:27:53) Intensity (green or red) indicates how much of the sample bound to each known sequence Can determine signal intensity for specific loci, whether RNA (gene transcription) or DNA (sequence copy number) Generating Transgenic Animals(00:27:53 - 00:28:28) Inject genes directly into a fertilized egg nucleus Problem: Eggs may have already started dividing, making it hard to target single-cell stage Often results in chimeric animals Embryonic Stem Cell Approach(00:28:28 - 00:29:14) Modify genes in embryonic stem cell samples Insert modified stem cells directly into a mouse embryo The stem cells will integrate and the resulting animal is a chimera Breeding Chimeric Animals(00:29:14 - 00:29:44) If the modified cells contribute to the germ line, the chimeric animal can be bred Offspring will inherit the genetic modification Utility of Chimeric Animals(00:29:44 - 00:30:19) Useful for studying lethal mutations that would be fatal in a full-body mutant The chimeric animal can produce offspring, some of which will have the lethal mutation Cloning(00:30:19 - 00:31:35) Remove the nucleus from an unfertilized egg Insert a nucleus from a somatic (body) cell of the animal to be cloned The egg will then develop into an embryo that is a genetic clone of the donor animal Challenges of Cloning(00:31:35 - 00:32:14) Early clones often died prematurely Thought to be due to improper epigenetic modifications in the somatic cell nucleus Techniques have improved to better reprogram the somatic cell nucleus Fluorescence Recovery After Photobleaching (FRAP)(00:32:14 - 00:32:55) Bleach a specific area of a fluorescently-labeled sample Measure how long it takes for fluorescence to "recover" as labeled molecules diffuse back into the bleached area Provides information about diffusion rates in the sample Fluorescence Lifetime Imaging Microscopy (FLIM)(00:33:13 - 00:33:39) Irradiate a cell with light and measure the fluorescence lifetime Can quantify concentrations of ions, molecules, and gases within the cell https://www.studyfetch.com/platform/studyset/66cd116dd279f5220d947c66/material/66ce6c0ee81bb65113fe36b3/document?go=note 8/8

Use Quizgecko on...
Browser
Browser