WSSP-Ch1Q-Vectors-MPS 2023 Molecular Cloning PDF

WSSP-23 Chapter 1 Vectors and Libraries  Today you will start doing something in the lab called "Molecular Cloning" Or "Genetic Engineering" Or "Recombinant DNA Technology" These techniques will al...

WSSP-23 Chapter 1 Vectors and Libraries  Today you will start doing something in the lab called "Molecular Cloning" Or "Genetic Engineering" Or "Recombinant DNA Technology" These techniques will allow you to study and manipulate individual genes Do Now What is a gene? A - a chromosome B - a protein C - part of a chromosome that is transcribed D - a section of DNA that codes for a protein E - is made of RNA For many years, biochemists had tried to purify genes. But they were frustrated because they are hard to purify.  Board Question: Why are genes hard to purify? a. Because they're tiny b. Because there are too few of them c. Because one piece of DNA looks like any other d. Because they are hard to separate from proteins e. Because they are too long Cloning Scheme Ligate Amplify and Prep p. 2-1 Cloning Scheme Insert and Vector are cut at sites A and B Digest Ligate Amplify and Prep p. 2-1 Cloning Scheme Insert and Vector are joined at sites A and B Ligate p. 2-1  Vector containing insert is transformed into E. coli for amplification and purification https://www.youtube.com/watch ?v=SdqJFA6mOkI&ab_channel Amplify and Prep =Addgene p. 2-1 Vectors In order to study a DNA fragment (e.g., a gene), it needs to be amplified and eventually purified. These tasks are accomplished by cloning the DNA into a vector. Vector: A vector is generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli (can be a virus or a plasmid). p. 1-1 BMW: But why use vectors? A - because passenger DNA won't replicate by itself B - because they're inexpensive C - because they're circular D - because they're small E - because passenger DNA is too big to get into cells by itself  Plasmids Plasmid: Circular DNA molecules found in bacteria Replicated by the host’s machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication. Some plasmids are present in E. coli at 200-500 copies/cell p. 1-4 Plasmid Engineering Plasmids also contain selectable markers. Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. https://www.youtube.com/watch ?v=GNMJBMtKKWU p. 1-4 Transforming plasmids into bacteria Very inefficient: less than 1/1000 cells are transformed with the circular plasmid (linear does not transform) p. 1-2 Transforming plasmids into bacteria Need to treat cells with Ca++ to transform plasmid Normal Cell (negatively charged membrane) Plasmid Ca++ Cell treated with CaCl2 (more postively charged membrane) Transforming plasmids into bacteria Very inefficient: less than 1/1000 cells are transformed with the plasmid How do you identify the few cells with the plasmid? Plasmid Engineering Plasmids also contain selectable markers. Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. p. 2-2 Plate cells on media with antibiotic Kills cells without the plasmid Cloning a DNA fragment Dead Cells Colony p. 1-2 Board Question: What is the most likely explanation if the plating of the ligation mix of the cDNA library transformation looked like this? A) The ligation did not work B) The cells were not competent C) The DNA was not added to the cells (Lawn of cells) D) The plates did not have antibiotic E) None of the above Board Question: What is the most likely explanation if the plating of the ligation mix of the cDNA library transformation looked like this? A) The ligation did not work (No cells) B) The cells were not competent C) The vector was degraded D) A, B or C E) None of the above Safety Features Modern cloning plasmids have been engineered so that they are incapable of transfer between bacterial cells Provide a level of biological containment. Naturally occurring plasmids with their associated drug resistance genes are responsible for the recent rise in antibiotic-resistant bacteria plaguing modern medicine. p. 1-3  Do Now Draw and identify the parts of a plasmid used for molecular cloning. https://www.youtube.com/watch?v=Y7gxELssMRw&ab_chan nel=PromegaCorporation LacZ gene codes for -galactosidase, (enzyme) X-gal (analog of lactose) Expression of LacZ can be used as a marker (screen) Screening for Inserts p. 1-3 BMW: What is a likely explanation if the transformation of the ligation looked like this? A) The ligation did not work B) The transformation did not work C) No DNA insert D) Forgot to add X-gal E) Forgot to add ampicillin Transform plasmid into bacteria Why a maximum of only one molecule per cell? A- Otherwise the cell will get too crowded B - A cell can't replicate two plasmids C - Cells don't like to have more than one plasmid in them D - It will be too confusing to have more than one plasmid in a cell E - Why not?  Do Now In a few sentences explain blue white selection. What does blue white selection screen for?  Do Now In bullet format, write the steps to create a genomic library! DNA Libraries DNA library - a random collection of DNA fragments from an organism cloned into a vector Ideally contains at least one copy of every DNA sequence. Easily maintained in the laboratory Can be manipulated in various ways to facilitate the isolation of a DNA fragment of interest to a scientist. Numerous types of libraries exist for various organisms - Genomic and cDNA. https://www.youtube.com/watch ?v=SvjeCxVu2dI p. 1-5 Construction and analysis of a genomic DNA library Shotgun sequencing p. 1-5 Construction and analysis of a genomic DNA library Construction and analysis of a genomic DNA library Construction and analysis of a genomic DNA library Want large clones to span the genomic DNA  Sequencing the human genome cost $3 billion. Efforts are being made to cut the cost of sequencing a specific human genome to $1,000 (or less) We are not sequencing a genomic DNA library. We are sequencing a cDNA library What's that and what is the difference between the two? A cDNA is a copy of RNA (usually mRNA) in the form of DNA.  mRNA is a processed RNA transcript (in eukaryotes). It is intended to be translated into a protein.  Construction of a cDNA library Why are there blue colonies? p. 1-6 Construction of a cDNA library Construction of a cDNA library ?  Do Now- 1. Take out your homework  2. What medium did we grown the ON culture in? 3. Explain what would happen if each part was removed from the solution. Purification of mRNA Collect and grind up plants in mild denaturing solution Spin out debris (Tissue, membranes, etc) Treat with DNAse (removes DNA) Treat with Phenol (removes protein) p. 1-8 Synthesis of cDNA from mRNA p. 1-8 SfiI digestion sites of pTRiplEX2 p. 1-9 Cloning Duckweed cDNA fragments into the pTriplEX2 polylinker cDNA Insert p. 1-10 Differences between a genomic and cDNA library Genomic Library cDNA Library Promoters Expressed genes Introns Transcription start sites Intergenic Open reading frames (ORFs) Non-expressed genes Splice points p.1-7 WSSP-17 Chapter 1B Plasmid Preps  # School Naming your clones 04. East Brunswick HS, NJ 06. Hillsborough HS, NJ 07. James Caldwell HS, NJ # JHU School 09. JP Stevens HS, NJ 34. Science & Math Acad. MD 11. Montville HS, NJ 35. Walter Johnson, MD 13. Pascack Hills HS, NJ 59. Col. Zadok Magruder HS, MD 14. Pascack Valley HS, NJ 62. Winston Churchill, MD 15. Rutgers Prep., NJ 81. South River HS, MD 17. The Pingry School 86Biotech Career Acad., MD Your initials Year 18. Watchung Hills HS, NJ 19. West Windsor-Plains. HSS, NJ 38. Hackettstown HS, NJ 47. Fair Lawn HS, NJ 49. Piscataway HS, NJ # LLNL School 50. The Frisch School, NJ 65. Dougherty HS, CA 64. Rae Kushner Yeshiva HS, NJ 66. Modesto HS, CA 70. Old Bridge HS, NJ 67. Tracy HS, CA 13AV12.20 81. South River HS, MD 68. Waipuhu HS, HI 90. Granada High School, CA 90. Granada High School, CA 94. Academy of Edison, NJ 106. Monte Vista HS, CA 95. Acad. Of Enrichment & Adv., NJ 96. Holmdel HS, NJ 97. Robbinsville, HS, NJ 104. Elmwood Park Memorial HS, NJ 105. North Brunswick Twnshp. HS, NJ 106. Monte Vista HS, CA 109. New Providence HS, NJ 110. Princeton HS, NJ School # Clone # 112. Kinnelon HS, NJ 113. Newark Academy 114. Toms River High School East 115. Toms River High School South 116. Hamburg Area High School 117. Columbia HS, NJ 118. Monroe Township High School 119. South Plainfield High School 120. Toms River HS North, NJ 121. Jefferson Twnsp HS, NJ 122. West Windsor-Plains. HSN, NJ 1. Grow the bacteria Grow an overnight (ON) culture of the desired bacteria in 2 ml of LB medium containing the ampicillin antibiotic for plasmid selection. Incubate the cultures at 37°C with vigorous shaking. amp p. 2-11  Clicker Question: Which of the following colonies would be best to pick for making an ON? A) D) B) E) C) © 2016 WSSP  Clicker Question: Why is it very important that you change toothpicks after each colony you pick to make an ON? A) Toothpicks are cheap. B) Cells from one colony would be mixed in an ON with cells from another colony. C) The liquid in the culture tube would contaminate the plate. D) It is easier to count how many clones you have done. E) It is not important to change toothpicks © 2016 WSSP Enter the names of the clones into the Google Docs Clone Report sheet  Which clone name is correct? A) 20WJ-1.17 B) 20-JM.1.17 C) 20JM-1-17 D) 20JM1.17 E) 20JM117 © 2016 WSSP Grown ON on Grown ON the bench at shaking at RT 37°C One way to tell if your ON is fully grown is to see if you can see writing if you hold the tube up to your notes Needs to Fully Grown grow longer 2a. Transfer the cells to a tube and centrifuge Transfer 1.5 ml of the culture to a microfuge tube and pellet the cells for 1 minute at full speed (12,000 rpm) in the microcentrifuge. First tap or gently vortex the glass culture tube to resuspend the cells which have settled. The culture can be transferred to the microfuge tube by pouring. (Follow steps in Lab 6) p. 1-12  2a. Centrifuge the samples Balance the tubes in the centrifuge Pellet the cells for 1 minute at full speed (10,000- 14,000 rpm) in the microcentrifuge.  2a. Centrifuge the samples Before After Make sure there is a good size pellet At this stage in the miniprep what do you want to keep? A) Supernatant B) Cell pellet C) Neither D)Both  2b. Remove the supernatant Remove the growth medium (supernatant) by pouring out into a waste cup. Leave the bacterial pellet as dry as possible so that additional solutions are not diluted. Supernatant Cell Pellet Pour off Blot on Towel Supernatant 3a. Resuspend the cell pellet Resuspend the bacterial pellet in 250 µl of P1 by pipeting up and down. Add 250 l of P1, cap the tube, and vortex on the highest setting (pipetman can be used). Look very closely for any undispersed pellet before proceeding to the next step. It is essential that the pellet be completely dispersed. P1 contains three essential components: glucose, Tris and EDTA. Glucose and Tris are used to buffer the pH of the cell suspension. EDTA is a chemical that chelates divalent cations (ions with charges of +2) in the suspension, such as Mg++. This helps break down the cell membrane and inactivate intracellular enzymes. p. 1-12 3. Resuspend the cell pellet in Buffer P1 Resuspend the bacterial pellet in 250 µl of Buffer P1 by pipeting up and down. NOTE: The volumes of the solutions used in the miniprep in the following slides are the ones for the Qiagen minipreps The volumes will be different if your using a GE kit or kit from another supplier 3. Resuspend the cell pellet in Buffer P1 ake sure the pellet is fully resuspended! Not fully Fully suspended suspended 4. Add Buffer P2 Add 250 µl of Buffer P2 (0.2 N NaOH, 1%SDS), mix gently 4-6 times. Do not vortex!! This will shear the DNA and contaminate your DNA preps. Denatures protein, DNA, RNA, membranes. During this step a viscous bacterial lysate forms (the cells lyse). p. 1-13 4. Add Buffer P2 (cont.) The cell solution should become clear Before After 5. Add Buffer N3 Add 350 µl of Buffer N3 (3 M KOAc, pH 4.8). Mix gently 4-6 times. Do not vortex. N3 neutralizes cell suspension. A white precipitate consisting of aggregated chromosomal DNA, RNA and cell debris and SDS will form. Plasmids will renature p. 1-13 5. Add Buffer N3 (cont.) Before Invertin Invertin Invertin g g g 3 times 6 times White 6. Centrifuge cell debris Centrifuge for 5 minutes at full speed in the microcentrifuge. A white pellet will form on the bottom and side of the tube after centrifugation. At this stage in the miniprep what do you want to keep? A) Supernatant B) Pellet C) Neither D) Both 7. Transfer sup. (DNA) to spin column. Pour the supernatant to the appropriately labeled spin column which has been inserted into the 2 ml microcentrifuge tube. 7. Transfer sup. (DNA) to spin column. Before After 8. Centrifuge the spin column Centrifuge for 1 minute at full speed. Before After At this stage in the miniprep what do you want to keep? A) Column B) Flow Through Column C) Neither D) Both Flow Through  DO NOW 1. List one FUN thing you did over break. 2. What is the purpose of the PCR? 3. If there is contamination in PCR, then you will not be sequenced. True or False. Explain.  DO NOW 1. What is the purpose of the miniprep? 2. List the steps we have already done of the miniprep. 3. List the remaining steps we discussed in class. 8. Centrifuge the spin column (cont.) Pour the flow-through from the collection tube. 9. Wash the column with Buffer PE Add 750 l of Buffer PE to the spin column contained in the 2 ml Collection Tube, centrifuge at full speed for 1 minute, and drain the flow through. This buffer helps to further remove any nucleases that may have co-purified with the DNA. Remove the liquid that has passed through the column in the same way as performed in Step 8. p. 1-14 9. Wash the column with Buffer PE Before After Spin At this stage in the miniprep what do you want to keep? A) Column B) Flow Through Column C) Neither D) Both Flow Through 10. Spin the column a second time to remove all the Buffer PE Centrifuge again for 1 minute at full speed to remove any residual wash solution that might still be in the column. Any residual wash solution must be removed because the ethanol contained in this solution may interfere with further DNA manipulations. abel a yellow tube for the plasmid DN Label the top with just your initials and clone # Label the side with the full clone name: School #, your initials, clone #, and year 11. Elute the DNA with EB Place the spin column into an appropriately labeled Yellow 1.7 ml microcentrifuge tube and add 50 ul of EB buffer to the column. Elutes the plasmid DNA from the column and collects in the microcentrifuge tube. p. 4-11 At this stage in the miniprep what do you want to keep? A) Column B) Flow Through Column C) Neither D) Both Flow Through 11. Elute the DNA with EB (cont.) Centrifuge at full speed for 1 minute. p. 1-15 12. Store your DNA Remove the spin column from the labeled 1.7 ml microcentrifuge tube and close the lid on the tube tightly. Store the miniprep DNA in your freezer box (-20C). p. 1-15 Board Question: Which of the following is the most serious error in the miniprep procedure? A) Not fully resuspending the cells in P1. B) Vortexing after N3 is added. C) Transferring some of the precipitate onto the column. D) Washing the column with Elution Buffer (EB) instead of PE. E) Eluting with 100 ul Elution Buffer (EB) instead of 50 ul.