7. BIOLOGY 510 RECOMBINANT DNA TECHNIQUES LABORATORY Lab: MW 2:00 – 4:50 p.m., 224 T.H. Morgan Lecture: F 2:00 – 2:50 p.m., 109 T.H. Morgan Instructor: Dr. Brian Rymond, 335A T.H. Morgan Building, 257 ‑ 5530, rymond@uky.edurymond@uky.edu Office hours Tuesday 9:15 ‑ 10:15 AM or by appointment Teaching Assistants: Chanung Wang (chanung.wang@uky.edu) and Swagata Ghosh (Swagata.ghosh@uky.edu)chanung.wang@uky.eduSwagata.ghosh@uky.edu Course Website:: https://bio.as.uky.edu/rymond/bio-510. This website contains a copy of the lab manual, Friday lectures PowerPoint slides, reading assignments, examples of old quizzes and exams, and class data. Check it frequently for updates.https://bio.as.uky.edu/rymond/bio-510 Course Content: This four-credit course familiarizes the advanced undergraduate and the beginning graduate student with the theory and practice of recombinant DNA technology and molecular genetic applications. Emphasis is placed on learning though direct experimentation. The laboratory techniques and skills acquired (e.g., DNA isolation and sub-cloning, prokaryotic/eukaryotic cell transformation, phenotypic selection of recombinant clones, RNAi knockdown, deep-sequencing validation, in vitro mutagenesis, protein purification, RNA analysis, real-time PCR, in vitro transcription ‑ among others) are broadly applicable in modern medical, industrial and basic biological research. The Friday lectures supplement the laboratory assignments with background information and descriptions of alternative or additional methodologies The Friday lectures generally will not involve discussion of the laboratory assignments or results. A PowerPoint file covering the Friday lectures will be posted on the class website and updated routinely throughout the semester.

8. Required Textbook & Background resources: BIO 510 is a reading intensive course. Principles of Gene Manipulation and Genomics, 7 th Edition S.B. Primrose, R.M. Twyman (Blackwell Science Press, 2006). This textbook provides a general overview of virtually all the techniques and approaches we use in the laboratory. It includes many scientific citations of historical interest and references relevant to technical innovations and other topical, directed reading. The text helps bring our exceptionally diverse enrollment up to a common level of understanding and is a useful starting point for our lectures and discussions. The textbook is not a lab manual and is not meant to explain the lab exercises. In addition, this textbook is not meant to replace the use of primary literature in your scientific education. Good books for background reading include GENES VII (B. Lewin, Oxford Press, 2000), Molecular Biology of the Cell (B. Alberts et al., Garland Press, 2002); Genomes (T.A. Brown, Garland Science, 2002), Biochemistry (Berg et al., W. H. Freeman & Co., 2002 and Modern Genetic Analysis (Griffiths et al., 1999). These and related books are searchable for free online at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=books. The Cold Spring Harbor Press web site (http://www.cshlpress.com/) is a particularly rich source of reference materials for students seeking to purchase professional lab manuals.W. H. Freeman & Co.Modern Genetic Analysis http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=bookshttp://www.cshlpress.com/

10. Joining DNA Ends with DNA Ligase Each Lab Member. NOTE: The volumes of vector and insert DNA may need to be altered depend­ing on your recovery so be sure to show the photograph of your gel to the instructor or T.A. before continu­ing. To increase the frequency of the bi-molecular ligation, we will use approximately 4X the molar amount of insert to vector. The insert is roughly 1/4 the length of the vector, so equal mass amounts of DNA (that is EtBr intensity bands) will give ~4X the molar amount of insert to vector. See pages 44-48 in PGMG. Assemble the reaction in the following order: 10 μl of sterile water (or sufficient water have a final reaction volume of 20 μl 2 μl of 10X ligase buffer * 2 μl (or as directed by the T.A.) of the pTZ18 or 19u Hind/Eco cut vector 5 μl (or as directed by the T.A.) of isolated gel fragment (i.e., insert DNA) Mix briefly, then add: 1 μl (1 to 2.5 units) of T4 DNA ligase. Mix briefly. ‑ Each student should also set up a second, control ligation reaction as above but with additional water instead of insert DNA. This control will score the amount of vector self-ligation. Here are several outstanding WEB sites for Molecular Biology protocols and tools (most web-based, some as free downloads): http://expasy.org/tools/ http://www.ncbi.nlm.nih.gov/Tools/index.html http://www.sanger.ac.uk/resources/software/ DICTIONARY OF MANY COMMON GENETIC AND MOLECULAR BIOLOGY TERMS: http://www.genome.gov/glossary/index.cfmhttp://www.genome.gov/glossary/index.cfm?

24. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase.

25. Each Student (read the pdf file on the Qiagen QIAquick spin handbook – gel recovery section) ‑ Mix 2 μl of dye (50% glycerol, 0.1% bromophenol blue, 0.1% xylene cyanol, 5X TAE) with 7.5 μl of your DNA digest in a clear microfuge tube. Pipet 4.5 µl of this DNA/dye mixture carefully into two wells of your 1.25 % agarose gel. Run at 70 volts until the fast dye (bromophenol blue) migrates ½ to 3/4 the gel length (NOTE: red electrode at the anode – farthest away from the well). ‑ With gloved hands remove the gel by lifting the glass slide (not the gel) on which it rests. Place the gel in the 2.0 μg/ml ethidium bromide solution. Ethidium bromide is mutagenic and a suspected carcinogen, take care not to get it on your skin. Go to our class web site to find information the structure of ethidium bromide and safe handling procedures. Also, read PGMG pages 16-17, 56. NOTE: To avoid microbial and nuclease contamination of your sample, close the lid of the pipet tip box after each use. CHANGES IN RED

26. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase.

27. pTZ18u, pTZ19u– made for high copy DNA amplification, RNA transcription, ssDNA production. IPTG X-gal The only difference between pTZ18u and pTZ19u is the orientation of the multiple cloning site with respect to the T7 RNA polymerase transcription site. 19u transcribes from HindIII to EcoRI and 18u transcribes from EcoRI to HindIII. Cloning in both vectors allows us to make both + and – strand RNA hybridization probes.

28. Each Student (groups 1-3 get pTZ18u and groups 4-6 get pTZ19u) Digest your pTZ vector (18u or 19u) with EcoRI in a final volume of 25 μl: 9.5 μl sterile water 2.5 μl 10X CutSmart buffer (1X = 50mM Potassium Acetate, 20mM Tris-acetate, 10mM Magnesium Acetate, 100μg/ml BSA, pH 7.9@25°C) 10 μl DNA (2 μg, ~ 1 picomole of 18u or 19u but not both) 1 μl EcoRI (or 10 units; excess units because our lab time is short & you want to ensure a complete digestion) 1 μl HinDIII Vortex the sample very briefly (~2 seconds) and then spin briefly (~5 seconds). Note that the air/liquid interface (foaming) can denature proteins, so limit the amount of vortexing time and avoid conditions that cause foaming. ‑ Incubate for 30min at 37 o C. ‑ Next, add 1 μl (0.3 unit) of shrimp alkaline phosphatase (SAP). Mix well then spin as before and then incubate at 37 o C for 15 min. For description of SAP see https://www.neb.com/products/m0371-shrimp-alkaline- phosphatase-rsap https://www.neb.com/products/m0371-shrimp-alkaline- phosphatase-rsap ‑ Add 150 μl of TE, extract 1X with 150 μl of PCI (phenol/CHCl 3 /isoamyl alcohol in a ratio of 50:49:1) by repeating the vortexing over a 5 minute period. Spin the sample in the microfuge for 5 minutes at full speed. Be careful, this organic solvent mix can burn your skin. ‑ Remove upper phase (aqueous, includes DNA) place into fresh microfuge tube. ‑ Add 100 μl of 3M sodium acetate and 1 ml of 100% ethanol, vortex. Label your tube with the date, your initials, and the experiment (i.e., pTZ18u E/H-SAP). Place in the -20 freezer until the next lab period. NOTE: In order for DNA or RNA precipitation to work, the sodium acetate and ethanol must be mixed well with your sample.

29. Dissolve & bind in QC (6M guanidine isothiocyanate, 50 mM Tris, 150 mM NaOAc, 20 mM EDTA pH 5 –WHY is pH relevant?), Wash in PE (80% ethanol in 10 mM Tris, pH 7.5) and Elute in EB (10 mM Tris, pH 8.5) Chaotropic salts such as 6M guanidinium isothiocyanate) interfere with the structure of water and increase the solubility of nonpolar compounds (purine & pyrimidine bases) in aqueous environments. Release occurs in low salt (EB) due to charge repulsion between DNA and silica resin. Bind DNA to the column, wash off impurities, elute DNA

30. The low pH keeps ionizable groups protonated and shields against charge repulsion between silica oxide and the DNA phosphate backbone.

31. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase. TODAY 3) Recover EcoRI/HindIII cut & SAP-treated pTZ18u (or 19u) DNA by ethanol precipitation & centrifugation. Score yield and integrity of this “vector” DNA and the recovered yeast “insert” DNA by agarose gel electrophoresis. Also, score the differences in DNA migration of the linear, circular and supercoiled forms of your plasmid DNA. 4) Prepare E. coli cells competent for DNA transformation by CaCl 2 treatment; cryopreserve the competent cells. Bonus: Comparing the sensitivity of EtBr and SYBR Safe for staining DNA in gels

33. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase. 3) Recover EcoRI/HindIII cut & SAP-treated pTZ18u (or 19u) DNA by ethanol precipitation & centrifugation. Score yield and integrity of this “vector” DNA and the recovered yeast “insert” DNA by agarose gel electrophoresis. Also, score the differences in DNA migration of the linear, circular and supercoiled forms of your plasmid DNA. 4) Prepare E. coli cells competent for DNA transformation by CaCl 2 treatment; cryopreserve the competent cells. Bonus: Comparing the sensitivity of EtBr and SYBR Safe for staining DNA in gels TODAY 5) Create the recombinant DNA molecule by directional cloning via ligation of EcoRI/HindIII cut plasmid vector (pTZ18u/19u) & yeast DNA insert. 6) Isolate yeast RNA to be used for the identification of cellular RNA transcripts originating from your yeast DNA insert. Bonus: Determine the influence of DNA buffer concentration on the mobility of linear and circular forms of ds DNA

34. EtBr appears more sensitive under the conditions used – although the manufacturer indicates similar sensitivity. May be possible to optimize with different filter sets. What about cost?

35. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase. 3) Recover EcoRI/HindIII cut & SAP-treated pTZ18u (or 19u) DNA by ethanol precipitation & centrifugation. Score yield and integrity of this “vector” DNA and the recovered yeast “insert” DNA by agarose gel electrophoresis. Also, score the differences in DNA migration of the linear, circular and supercoiled forms of your plasmid DNA. 4) Prepare E. coli cells competent for DNA transformation by CaCl 2 treatment; cryopreserve the competent cells. Bonus: Comparing the sensitivity of EtBr and SYBR Safe for staining DNA in gels. 5) Create the recombinant DNA molecule by directional cloning via ligation of EcoRI/HindIII cut plasmid vector (pTZ18u/19u) & yeast DNA insert. 6) Isolate yeast RNA to be used for the identification of cellular RNA transcripts originating from your yeast DNA insert. Bonus: Determine the influence of DNA buffer concentration on the mobility of linear and circular forms of ds DNA TODAY 7. Transform your competent E. coli with your experimental & control ligation reactions. Select transformants on LB-ampicillin plates and score putative recombinant transformants as β galactosidase deficient with IPTG/Xgal. 8. Determine yeast mRNA yield & prep 15 µg for your northern blot

36. Cost per standard mini-gel EtBr (purchased as powder): $0.0037/gel Sybr Safe (purchased as liquid concentrate):$2.45/gel Sybr safe is roughly 662 X more expensive to use when considering only the initial product cost (does not consider the possibility of re- using the solutions, possible different costs of disposal, etc.)

37. Basics of Gel Electric Circuits Amperage (amps for short) is a measure of electrical current the AMOUNT of electricity used. Voltage (volts) measures the pressure, or FORCE, of electricity. The amps multiplied by the volts gives you the wattage (watts), a measure POWER or the work that electricity does per second. http://www.helcohi.com/sse/body/hp.html http://www.helcohi.com/sse/body/hp.html V (volts) = I (milliamps) X R (resistance; measured in ohms.)ohms For a segment of a gel/buffer system –  cross-sectional area of buffer or gel,  resistance –  strength of buffer = [ion],  resistance – most resistance is in the agarose gel itself

38. Where does the current come from? Ions supplied by the buffer The charge on the macromolecules being separated Electrolysis of water – 4H 2 O  2H 2 + O 2 + 2H 2 O – self-ionization of water throughout the buffer: 4H 2 0  4H + + 4OH - At the negative pole – 4H + + 4e -  2H 2 At the positive pole – 4OH -  O 2 + 2H 2 O + 4e - – At which electrode would you expect more bubbles? Why?

39. Where does the current come from? Electrolysis of water – 4H 2 O  2H 2 + O 2 + 2H 2 O – self-ionization of water throughout the buffer: 4H 2 0  4H + + 4OH - At the negative pole – 4H + + 4e -  2H 2 At the positive pole – 4OH -  O 2 + 2H 2 O + 4e - – At which electrode would you expect more bubbles? Why?

41. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase. 3) Recover EcoRI/HindIII cut & SAP-treated pTZ18u (or 19u) DNA by ethanol precipitation & centrifugation. Score yield and integrity of this “vector” DNA and the recovered yeast “insert” DNA by agarose gel electrophoresis. Also, score the differences in DNA migration of the linear, circular and supercoiled forms of your plasmid DNA. 4) Prepare E. coli cells competent for DNA transformation by CaCl 2 treatment; cryopreserve the competent cells. Bonus: Comparing the sensitivity of EtBr and SYBR Safe for staining DNA in gels. 5) Create the recombinant DNA molecule by directional cloning via ligation of EcoRI/HindIII cut plasmid vector (pTZ18u/19u) & yeast DNA insert. 6) Isolate yeast RNA to be used for the identification of cellular RNA transcripts originating from your yeast DNA insert. Bonus: Determine the influence of DNA buffer concentration on the mobility of linear and circular forms of ds DNA 7. Transform your competent E. coli with your experimental & control ligation reactions. Select transformants on LB-ampicillin plates and score putative recombinant transformants as β galactosidase deficient with IPTG/Xgal. 8. Determine yeast mRNA yield & prep 15 µg for your northern blot TODAY 9. Resolve yeast total cellular RNA (nuclear, cytoplasmic, mitochondrial, poly A+/-) by denaturing gel electrophoresis (1% agarose, formaldehyde gel run in MOPS buffer). Transfer to positively charged nylon membrane (Millipore Immobilon NY + ). 10. Count blue & white colonies & patch single isolates from your experimental (pTZ18/19u + insert) and control (pTZ18/19u only) transformations. EXTRA – Onsite radiation training

42. All colonies – blue and white are transformants since only cells containing a plasmid grow on ampicillin. Blue colonies produce  – galactosidase and retain intact lacZ gene and likely represent the starting (unmodified) pTZ18u/19u. While colonies from the experimental plate are candidates for having recombinant plasmids with the “insert” DNA disrupting and inactivating lacZ

43. Satellite colonies are small colonies of non- transformed E. coli that surround an authentic plasmid transformant. Satellite colonies form when the local ampicillin concentration drops below a bacteriostatic level due to the b lactamase secreted by the large transformed cell. Satellite colonies do not have a drug-resistant plasmid and will not grow if re-streaked on a fresh ampicillin plate. Do not include the satellite colonies as “white” colonies on today’s plates.

44. NORTHERN BLOT: The gel-resolved yeast RNA will be transferred to our Immobilon NY+ positively charged membrane by capillary action. The image above shows the order of materials. We will do the transfer directly on the bench – starting with the Saran wrap (plastic wrap) but not include the “platform”. The gel and the lower Whatman (blotting) paper will be saturated with the 10X SSC transfer and provide the mobile liquid phase which carries the RNA upward where it sticks to the Immobilion membrane. After transfer, Chanung will photograph the transfer with a ruler & then covalently crosslink the RNA to the membrane with UV light followed by baking at 80C.

45. We will use the very abundant yeast 25S rRNA (3700 nts) and 18S rRNA (1700 nts) as internal molecular weight markers to create a standard curve to estimate the length of any yeast RNA that hybridizes to our probe. Nucleic Acids Res.Nucleic Acids Res. 2010, 38(4):1261-72. The Svedberg Unit (S) As ribosomal particles were first isolated from cell lysates by ultracentrifugation, the ribosomes and their sub-particles were named according to their sedimentation characteristics during centrifugation. The sedimentation properties of a particle depends on its molecular size and geometrical shape. The sedimentation characteristics also depend on the physical properties of the solution through which the particle is sedimenting. The two eukaryotic ribosomal subunits are referred to as the 40S and the 60S ribosomal subunits. The protein-free yeast large/small subunit rRNAs are 25S and 18S respectively.

46. Homework Example Outline Organism: Saccharomyces cerevisiae Vector: YCplac33 Type: ds DNA plasmid Selectable marker gene for yeast: URA3. This marker gene encodes the enzyme orotidine 5'-phosphate decarboxylase which is required for uracil biosynthesis in yeast. Plasmid selection is done in yeast that have a mutation in the chromosomal ura3 gene and the transformants are selected on medium that lacks uracil.rotidine 5'-phosphate decarboxylase Citation: Construction of ploidy series of Saccharomyces cerevisiae by the plasmid YCplac33-GHK. Hou L, Li X, Wang C, Cao X, Wang H.Construction of ploidy series of Saccharomyces cerevisiae by the plasmid YCplac33-GHK. J Ind Microbiol Biotechnol. 2013 Apr;40(3-4):393-7 Selection vs Screen?

50. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase. 3) Recover EcoRI/HindIII cut & SAP-treated pTZ18u (or 19u) DNA by ethanol precipitation & centrifugation. Score yield and integrity of this “vector” DNA and the recovered yeast “insert” DNA by agarose gel electrophoresis. Also, score the differences in DNA migration of the linear, circular and supercoiled forms of your plasmid DNA. 4) Prepare E. coli cells competent for DNA transformation by CaCl 2 treatment; cryopreserve the competent cells. Bonus: Comparing the sensitivity of EtBr and SYBR Safe for staining DNA in gels. 5) Create the recombinant DNA molecule by directional cloning via ligation of EcoRI/HindIII cut plasmid vector (pTZ18u/19u) & yeast DNA insert. 6) Isolate yeast RNA to be used for the identification of cellular RNA transcripts originating from your yeast DNA insert. Bonus: Determine the influence of DNA buffer concentration on the mobility of linear and circular forms of ds DNA 7. Transform your competent E. coli with your experimental & control ligation reactions. Select transformants on LB-ampicillin plates and score putative recombinant transformants as β galactosidase deficient with IPTG/Xgal. 8. Determine yeast mRNA yield & prep 15 µg for your northern blot 9. Resolve yeast total cellular RNA (nuclear, cytoplasmic, mitochondrial, poly A+/-) by denaturing gel electrophoresis (1% agarose, formaldehyde gel run in MOPS buffer). Transfer to positively charged nylon membrane (Millipore Immobilon NY + ). 10. Count blue & white colonies & patch single isolates from your experimental (pTZ18/19u + insert) and control (pTZ18/19u only) transformations. EXTRA – Onsite radiation training TODAY 11. Extract and analyze plasmid DNA from the white & blue colony transformants 12. Prehybridization of the northern blot of yeast cellular RNA

51. Homework 4 Question 1. Estimated length of yeast DNA (“insert” DNA): 650 bp Mass in Daltons = 650 bp X 650 Da/bp = ~422,500 Da (423 kDa) # molecules (from NEB catalog) 1 ug of 1000 bp fragment = 9.1 X 10 11 molecules We have 4 ug of a 650 bp DNA fragment, so 9.1 X 10 11 X 4 X 1000/650 = 5.6 X 10 12 molecules or using Avogadro's number of 6.022 X 10 23 molecules/mole where 422,500 gm of the 650 bp fragment = 1 mole 422,500 gm/1 mole = 4 X 10-6 g/X mole = 9.467 X 10 -12 mole 6.022 X 10 23 molecules/mole = X molecules/9.467 X 10 -12 mole X= 5.7 X 10 12 molecules # of 5’ ends is twice the number of ds DNA molecules (since each strand has one 5’ end) So, 5.6 X 10 12 molecules X 2 = 1.12 X 10 13 5’ ends in 4 ug of your 650 bp insert DNA

52. Question 2: What is a neoschizomer? (2 points). Two different restriction endonucleases that share the same recognition sequence but cut at two different positions within the sequence. Example from NEB book AatII (recognition sequence: GACGT↓C) and ZraI (recognition sequence: GAC↓GTC) Question 3: The genetic code is NOT actually universal. Provide two different examples where a specific codon (group of 3 nucleotides) defines different genetic information than what is produced by nuclear genes that mRNA. List the codon, state what is encoded in the standard and the atypical genetic code, and state the organism and genome (e.g., nuclear, chloroplast, mitochondrial) involved (5 points). See: http://www.ncbi.nlm.nih.gov/Taxo nomy/Utils/wprintgc.cgi http://www.ncbi.nlm.nih.gov/Taxo nomy/Utils/wprintgc.cgi For an extensive list of exceptions

53. “Mini-prep” solutions to extract plasmid DNA from E. coli Solution I. 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0). This resuspends the pellet in an osmotically stabilized buffered solution where EDTA helps inhibit divalent cation-dependent nucleases. The pellets must be dissolved completely. Hold the tube up to the light and roll the liquid around to determine if any “clumps” of cells are present. If need be, use a P-200 tip to disrupt the clumps. Solution II. (200 mM NaOH, 1% SDS). This high pH, strong ionic detergent lysis the cells. Mix by rapid inversion 5 times; do not vortex. Incubate at room temperature for 3 minutes. 3. Solution III. (3M NaOAc made by mixing 300 ml of 5M potassium acetate with 57.5 ml of glacial acetic acid and 142.5 ml of sterile water). This high-salt solution neutralizes the pH and causes proteins and chromosomal DNA (but not plasmid) to precipitate. Mix by rapid inversion 5 times; do not vortex. Next, PCI & chloroform extractions followed by ethanol precipitation.

54. Northern blot prehybridization At this time, a print of your cellular RNAs are covalently bound to the surface of the positively charged nylon membrane. The next step is to block all remaining free surface (i.e., not bound with your RNA) on the nylon membrane with a non-specific nucleic acid. Why should we do this? The blocking step is done with heat denatured salmon sperm DNA in a solution composed of simple salts that (6X SSC) and detergents and organic compounds that promote nucleic acid/membrane interactions (Denhardts). Why do you think salmon sperm DNA is used? We will wrap the membrane in synthetic nylon mesh and place this in a glass roller bottle which will rotate to distribute the blocking solution over the northern blot surface. Be sure that the roller bottle is tightly capped.

55. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase. 3) Recover EcoRI/HindIII cut & SAP-treated pTZ18u (or 19u) DNA by ethanol precipitation & centrifugation. Score yield and integrity of this “vector” DNA and the recovered yeast “insert” DNA by agarose gel electrophoresis. Also, score the differences in DNA migration of the linear, circular and supercoiled forms of your plasmid DNA. 4) Prepare E. coli cells competent for DNA transformation by CaCl 2 treatment; cryopreserve the competent cells. Bonus: Comparing the sensitivity of EtBr and SYBR Safe for staining DNA in gels. 5) Create the recombinant DNA molecule by directional cloning via ligation of EcoRI/HindIII cut plasmid vector (pTZ18u/19u) & yeast DNA insert. 6) Isolate yeast RNA to be used for the identification of cellular RNA transcripts originating from your yeast DNA insert. Bonus: Determine the influence of DNA buffer concentration on the mobility of linear and circular forms of ds DNA 7. Transform your competent E. coli with your experimental & control ligation reactions. Select transformants on LB-ampicillin plates and score putative recombinant transformants as β galactosidase deficient with IPTG/Xgal. 8. Determine yeast mRNA yield & prep 15 µg for your northern blot 9. Resolve yeast total cellular RNA (nuclear, cytoplasmic, mitochondrial, poly A+/-) by denaturing gel electrophoresis (1% agarose, formaldehyde gel run in MOPS buffer). Transfer to positively charged nylon membrane (Millipore Immobilon NY + ). 10. Count blue & white colonies & patch single isolates from your experimental (pTZ18/19u + insert) and control (pTZ18/19u only) transformations. EXTRA – Onsite radiation training 11. Extract and analyze plasmid DNA from the white & blue colony transformants 12. Prehybridization of the northern blot of yeast cellular RNA TODAY 13. Prepare recombinant DNA for in vitro transcription (linearize plasmid downstream of insert). Store pre-hybridized membrane until use next Monday.

56. Running list of experiments Goals: Use of PCR to a) identify gene variants, b) validate mRNA deep sequencing results, and c) measure nucleic acid abundance Today 1. Identify gene variants. Isolate genomic DNA from haploid yeast strains with 1) a wildtype SQS1 gene 2) a sqs1::KAN gene disruption and from a diploid strain with both at SQS1 and sqs1::KAN allele.

57. Gel shift is obviousNo shift obvious (some DNA 1 kbp marker spillover in lane 2) Most result were pretty good in DNA yield and recovery of the desired recombinant DNA. Why does the plasmid DNA run as a series of bands rather than as a single band?

58. http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=0CC4QFjACahUKEwjoiaegwo3IAhXLzYAKHZaXBA4&url=h ttp%3A%2F%2Fmmbr.asm.org%2Fcontent%2F62%2F2%2F434.full&usg=AFQjCNHson7VoJUQ3DRkQ04Fr7lcd0ImDA Web image showing similar banding patterns (look at last lane only). Dimers and other multimers form by plasmid recombination. We also sometimes see small amounts of slowly migrating E. coli DNA in the area.

59. Chromosomal and plasmid DNA in living bacteria is negatively supercoiled (~ 1 negative supercoil per 40 turns of the helix). DNA gyrase is a topoisomerase II enzyme that uses ATP to add negative supercoils (to relax the positive supercoils produced during replication & transcription) –it introduces double- stranded DNA breaks followed by gyrase-mediated re-ligation that can help resolve (or form) plasmid dimers (mainly a function of topoisomerase IV). DNA gyrase facilitates the replication of DNA because the negative coils neutralize the positive supercoils introduced during opening of the double helix by DNA polymerase and helicase. https://www.youtube.com/watch?v=wqoeVrsbPn8 https://www.youtube.com/watch?v=T06lo8T8Pmw Topoisomerase I is a competing enzymatic activity that relaxes negative (or positive) supercoils it; it produces a single stranded nick in one strand to unwind and then reseals. Removes supercoiling to enhance separation of DNA strands upon completion of replication. Topo I activity (E. coli TopA gene product) does not require ATP.

60. Electron micrograph of different levels of supercoiling of plasmid DNA. http://chemistry.umeche.maine.edu/CHY431/Nucleic5.html http://chemistry.umeche.maine.edu/CHY431/Nucleic5.html

61. Progressive relaxation of supercoiled DNA by topoisomerase activity. PNAS, 93:14416– 14421 (1996) Novobiocin is an inhibitor of this topoisomerase. Some cancer drugs target topoisomerase – why might this be a good strategy in cancer? Topoisomerase I inhibitors: irinotecan, topotecan, camptothecin and lamellarin D all target type IB topoisomerases, Topoisomerase II inhibitors: etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid,[5] and HU-331, a quinolone synthesized from cannabidiol.

62. Running list of experiments Goal: Characterization of a Eukaryotic Gene Steps: Cloning a segment of Saccharomyces cerevisiae (baker’s yeast) DNA into a pair of convenient plasmid vectors, specifically, pTZ18u and pTZ19u. 1) Gel fractionate the EcoRI/HindIII cleaved yeast DNA and recover the yeast DNA band free of a second DNA band (originating from another plasmid, pUC19 which lacks some of the desired features of pTZ18u & pTZ19u). 2) Prepare the pTZ18/19u plasmids for directional DNA cloning but cutting each plasmid at its multiple cloning site with two different restriction endonucleases, EcoRI and HindIII. To ensure that the any singly-cleaved plasmid DNAs do not “self ligate” (which will increase the background of non-recombinant clones subsequently recovered) we will remove the 5’ phosphate from the vector DNA with shrimp alkaline phosphatase. 3) Recover EcoRI/HindIII cut & SAP-treated pTZ18u (or 19u) DNA by ethanol precipitation & centrifugation. Score yield and integrity of this “vector” DNA and the recovered yeast “insert” DNA by agarose gel electrophoresis. Also, score the differences in DNA migration of the linear, circular and supercoiled forms of your plasmid DNA. 4) Prepare E. coli cells competent for DNA transformation by CaCl 2 treatment; cryopreserve the competent cells. Bonus: Comparing the sensitivity of EtBr and SYBR Safe for staining DNA in gels. 5) Create the recombinant DNA molecule by directional cloning via ligation of EcoRI/HindIII cut plasmid vector (pTZ18u/19u) & yeast DNA insert. 6) Isolate yeast RNA to be used for the identification of cellular RNA transcripts originating from your yeast DNA insert. Bonus: Determine the influence of DNA buffer concentration on the mobility of linear and circular forms of ds DNA 7. Transform your competent E. coli with your experimental & control ligation reactions. Select transformants on LB-ampicillin plates and score putative recombinant transformants as β galactosidase deficient with IPTG/Xgal. 8. Determine yeast mRNA yield & prep 15 µg for your northern blot 9. Resolve yeast total cellular RNA (nuclear, cytoplasmic, mitochondrial, poly A+/-) by denaturing gel electrophoresis (1% agarose, formaldehyde gel run in MOPS buffer). Transfer to positively charged nylon membrane (Millipore Immobilon NY + ). 10. Count blue & white colonies & patch single isolates from your experimental (pTZ18/19u + insert) and control (pTZ18/19u only) transformations. EXTRA – Onsite radiation training 11. Extract and analyze plasmid DNA from the white & blue colony transformants 12. Prehybridization of the northern blot of yeast cellular RNA 13. Prepare recombinant DNA for in vitro transcription (linearize plasmid downstream of insert). Store pre-hybridized membrane until use next Monday. TODAY 14. In vitro transcription of 32 P-labeled plus (+) and minus (-) strand DNA probes with T7 RNA polymerase 15. Separation of unincorporated 32 P-UTP from the probe by size exclusion chromatography 16. Hybridization of each groups blot with radiolabled RNA (riboprobe) transcribed from the yeast DNA insert of the recombinant pTZ18u and pTZ19u plasmids

63. Good cleavage & yield. Lanes (L-R): Marker, uncut, Cut (1), Cut(2) Incomplete cleavage (set 1) or yield (set 2). Lanes (L-R): uncut, Cut (1), Marker, uncut, Cut (2), Cut(2), ?

64. Scintillation counter – beta particle emissions from the radioactivity absorbed by scintillation cocktail which emits a secondary light photon identified by the photomultiplier tube and converted into electrical signals which are presented as counts per minute. How well the machine “sees/records/senses” the radioactivity depends on the particular isotope and on the machine used

65. Sequence alignment of human and Zebrafish rhodopsin

66. 1) Transformation Efficiency: 130 white + 20 blue = 150 transformants on 250 µl plate. 0.1µg of plasmid DNA in the whole ~2.5 ml of transformation – so, the 250 µl plate received ~0.01 µg of plasmid DNA 150 transformants/0.01 µg = 15,000 transformants per microgram 2) % viable cells transformed 3.7 X 10 8 cells per ml X 0.25 ml = 9.25 X 10 7 viable cells on the 250 µl plate where 150 colonies (transformants) were present 150 transformants/ 9.25 X 10 7 viable cells X 100 = 1.62 X 10 -4 = 0.000162% 3) What’s limiting for transformation? Two experiments – first, use the same amount of cells used in our lab but vary the DNA amount (e.g., 0.01 µg, 0.1 µg, 1.0 µg) in three parallel transformations. Second experiment: Use the same amount of DNA used in our lab, but vary cell concentration (e.g., 1X, 1/3X, and 3X) in three parallel transformations. 4) % Recombinant transformants = 130/150 X 100 = 86.7% 5) Uncut non-recombinant vector expected to produce blue colonies.

67. 6. Blue colonies on experimental plate, blue or white on the control plate. We used a forced ligation with two different restriction enzymes with incompatible ends plus dephosphorylated the 5’ ends of the vector to prevent re-formation of the original (non-recombinant) plasmid expected to be the blue transformants. As such, the blue transformants likely arose from plasmids either not cut by either restriction enzyme or cut by one enzyme and not dephosphorylated so that the circle was re-formed by ligation. Also, it is possible that if both HinDIII and EcoRI cut, but the phosphatase was inefficient, the small released HinDIII EcoRI band may have re-inserted into the vector, reforming the original pTZ18u/19u. Blue colonies on the while plate had the same origin. The while colonies most likely come from linear non-recombinant pTZ18u/19u that entered the E. coli, was trimmed by exonuclease that removed part of lacZ, and then religated with E. coli DNA ligase to form a smaller deletion derivative of the original non-recombinant pTZ18u/19u.