1. Molecular Biology II Molecular cloning and mouse knockout principles

2. 1- Molecular cloning

3. The tools that fuel molecular cloning Restriction digest of DNA DNA ligation PCR https://www.neb.com/products/restriction-endonucleases

4. The Nobel Prize in Physiology or Medicine 1978 jointly to Werner Arber (Switzerland), Daniel Nathans (USA) and Hamilton O. Smith (USA) for the discovery of restriction enzymes and their application to problems of molecular genetics.

5. The tools that fuel molecular cloning Restriction digest of DNA DNA ligation PCR https://www.neb.com/products/restriction-endonucleases

6. The Nobel Prize in Chemistry 1993 Michael Smith (Canada), for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies Kary B. Mullis (USA), for his invention of the polymerase chain reaction (PCR) method

7. Restriction Endonucleases

8. Found in Bacteria or Archaea Cleave DNA depending on the presence of specific recognition sites (vary from short palindromyc sequences like GAATTC to long asymmetrical sequences like TGAN 8 TGCT) Do not cleave DNA of the producing organism, which is protected by methylation (there are a few exceptions) Part of a simple bacterial “immune system” protecting from foreign DNA (bacteriophages) Restriction Endonucleases

9. Sites of methylation in bacteria, recognized by restriction endonucleases http://www.mikeblaber.org/oldwine Restriction Endonucleases

10. Type I: cuts the DNA at a random site far (>1000 bp) from the long asymmetrical recognition sequence. Consists of 3 different subunits (for DNA modification, cleavage and sequence recognition). Requires ATP. Restriction Endonucleases Type II: cuts the DNA within or near the recognition sequence (4-8 bp). Usially homodimers (only DNA cleavage and sequence recognition; no methyltransferase activity). Does not require ATP.

11. Type III: cuts the DNA about 20-25 base pairs from the recognition sequence (5-7 bp). Consists of 2 different subunits (for DNA recognition/modification and cleavage). Requires ATP. Restriction Endonucleases Type IV: cuts modified (e.g. methylated) DNA at a random site. Consists of 2 different subunits (for DNA recognition and cleavage). Requires GTP.

12. More than 4,000 restriction endonucleases are known, recognizing more than 300 distinct sequences (see REBASE® at rebase.neb.com) REBASE® lists (October 2004) only 59 Type I, 10 Type III and 3 Type IV restriction endonucleases (sequence analysis predicts approximately 29% Type I, 45% Type II, 8% Type III and 18% Type IV) Thus, in common usage, the term “restriction enzyme” is usually identified with the restriction endonucleases of type II Restriction Endonucleases

13. Type II Restriction Endonucleases Used widely as a tool to manipulate DNA sequences Most useful for cloning are enzymes with six or eight bp palindromyc recognition sites http://www.bris.ac.uk/bioche mistry/halford/research.html

14. Naming of Restriction Endonucleases The first three letters refer to the organism from which the restriction enzyme was originally isolated The fourth letter (if present) refers to the strain The Roman numerals serve as indices if the same organism contains several different restriction enzymes. BamH I: Bacillus amyloliquefaciens, strain H, enzyme I Hind III: Haemophilus influenzae strain d, enzyme III

15. Type II Restriction Endonucleases Wikipedia Crystal structure of EcoRI bound to DNA

16. Frequency of restriction sites If the base composition is assumed to be completely random, the restriction site frequency can be calculated as 4 x, with X being the length of the recognition sequence For a four-base cutter: 4 4 = 256  one cutting site every 256 bp “ “ six “ “ : 4 6 = 4096  one cutting site every 4096 bp However, base compositions are not completely random and strongly depend on the organism, and hence so does the frequency of restriction sites.

18. Restriction digests Preparative digest (to obtain DNA fragments for cloning; 100-500 ng DNA) –≥ 20 units of enzyme(s), max 10% of total reaction volume (20-50 ul) (really, you may use much more considering the enzyme quality, especially when cutting the vector to which you ligate your DNA) –>1 hour incubation Analytical digest (to verify construct) –5 units of enzyme, reaction volume as little as 10 ul –1 hour incubation

19. General practice for restriction digests Aliquot your buffers –Sharing is fun, but it is not a good idea to have ten people dip into the same solutions for their digests, as this increases the likelihood of contaminations Prepare digests in a clean working environment –Work on a clean bench –You may want to consider wearing gloves

21. Know your enzyme! “Standard” conditions work “usually”, but depending on the enzyme used, you will have to pay attention to specific features A wide variety of information is available for you from the manufacturer’s websites – but how do you read/interpret these properly? – what information is important for you?

22. An example: New England Biolabs Enzyme descriptions No, this is not an advertisement!!!! Enzymes from other providers (Roche, Fermentas) are very good too! Quick overview of enzyme features Cloned at NEB, Inc. Purified from a recombinant sourse Can digest 1 µg DNA in 5 min Passed the blue-white selection Heat inactivation possible

23. What is a “unit”? Restriction Enzyme Unit Definition: “One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (~50 000 bp) in 1 hour at 37°C in a total reaction volume of 50 µl”. An example: New England Biolabs Enzyme descriptions Enzyme Unit (in general): One unit is defined as the amount of enzyme that catalyzes the conversion of 1 micro mole of substrate per minute.

24. The unit definition refers to cleavage of linear (phage λ) DNA But: Plasmid DNA is usually supercoiled! * * More about this later!

25. The recognition site and cutting features NcoI produces “sticky” ends with a 4 base 5’ overhang Example for “blunt” end cutting: the PvuII cutting site

26. Source of enzyme

27. Nocardia (yellow) Wikipedia American Type Culture Collection Application Oxidizes alkylbenzenes Produces restriction endonuclease NcoI Isolation Soil Type Strain no Biosafety Level 1 Product Format freeze-dried Price€427.00

28. Reaction buffer features 1X NEBuffer 3: 100 mM NaCl 50 mM Tris-HCl 10 mM MgCl 2 1 mM Dithiothreitol pH 7.9 @ 25°C 1X NEBuffer 2: 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl 2 1 mM Dithiothreitol pH 7.9 @ 25°C 1X NEBuffer 4: 50 mM potassium acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 1 mM Dithiothreitol pH 7.9 @ 25°C 1X NEBuffer 1: 10 mM Bis-Tris- Propane-HCl 10 mM MgCl 2 1 mM Dithiothreitol pH 7.0 @ 25°C Very low salt, Low pH Low saltHigh salt Low salt, Acetate buffer Buffer compatibility

29. Some enzymes require special buffers Sometimes additives are required BSA = bovine serum albumin SAM = S-adenosylmethionine Using the wrong buffer or leaving out required additives can result in: –Decreased or no activity (pH too low or too high; salt too high; omission of BSA or SAM) –Star activity (loss of cutting specificity) Digests with two enzymes sometimes may require compromises in buffer usage

30. What is STAR activity? Some enzymes lose specificity if they are used in the wrong buffer conditions. Instead of recognizing a six base-pair sequence, the enzymes are able to cleave four base- pair sites Example: Eco RI restriction site is GAATTC EcoRI STAR sites are AATT  EcoRI becomes a four-base cutter!

31. STAR activity http://www.fermentas.com/techinfo/re/restrstaract.htm

32. What conditions allow for STAR activity (and how is it prevented)? Low salt (  use salt concentrations >100mM) Low pH (  use pH of about 8.0) Glycerol concentration higher than 5% (  the fraction of restriction enzyme preparation should not exceed 10% of the total digest volume) High enzyme concentrations (  don’t use excessive amounts)

33. Ligation and recutting: Enzyme Quality information Information about exonuclease activity in the enzyme preparation. “Good” enzymes allow up to 200- fold overdigestion! (i.e. there is very little exonuclease activity in the preparation) http://www.fastbleep.com

34. Other information - Concentration of enzyme in the form sold by NEB -Storage conditions (buffer in which the enzyme is supplied) -Compatibility with buffers for diluted stocks of the enzyme -Heat inactivation (may be important in double digests with buffer incompatibilities or if cutting after a ligation reaction).

35. About dam, dcm and CpG methylation dam and dcm methylation is carried out by certain E. coli strains and this DNA modification can interfere with restriction enzyme function –Dam methylase modifies the adenine in the sequence GATC –Dcm methylase “ “ cytosine in the sequences CCAGG and CCTGG Only DNA isolated from dam+ or dcm+ bacterial strains will be modified; PCR products do not carry the modification dam or dcm methylation can be reversed by passing vectors through dam- or dcm- E. coli strains

36. About dam, dcm and CpG methylation CpG methylation of cytosine takes place in eukaryotic tissues Only DNA isolated from eukaryotic sources (like genomic DNA) may carry CpG methylated sites; once the DNA is passaged in a bacterial host, the DNA modification is not maintained, and also PCR products will not carry the methylation http://clincancerres.aacrjournals.org/content/11/18/6409/F1.expansion

37. Cleavage of supercoiled DNA Enzyme units are usually given for digests of linear DNA  works fine for PCR products Plasmids are circular and usually supercoiled Biotechnology and Applied Biochemistry (2007) 46, (97–103) Supercoiled form Relaxed form

38. Cleavage of supercoiled DNA Some enzymes cleave supercoiled DNA at much lower rates For some enzymes, reduction in activity can be as high as 10-fold! Problem if only one enzyme is used, or if both enzymes in a double digest are inefficient in cleaving supercoiled DNA (e.g. SalI and NheI)  add more enzyme

39. Cleavage close to ends Enzymes may need a bit of extra DNA to “sit” on in addition to their recognition sequence This becomes important when cutting at the end of DNA sequences, e.g. PCR products or sequences in close proximity in a multiple cloning site (MCS)  add the appropriate amount of extra bases to your PCR primer if introducing restriction sites to the ends of DNA sequences (four or more?)  avoid choosing restriction sites that are adjacent in an MCS –If you absolutely have no other choice, try using in the mixture of 2 restriction endonucleases first the enzyme that needs extra adjacent bases and then an enzyme that tolerates proximity to ends

40. Example: HindIII requires three extra bases for good cutting, SphI only needs one extra base  add HindIII first, then (after 30-60 minutes) add SphI (check https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the- end-of-dna-fragments)

41. Keep in mind! There are also: Isoschizomers –Enzymes that have the same recognition sequence Neoschizomers –Enzymes that have the same recognition sequence, but cleave in a different position AatII (recognition sequence: GACGT↓C) and ZraI (recognition sequence: GAC↓GTC) are neoschizomers of one another, while HpaII (recognition sequence: C↓CGG) and MspI (recognition sequence: C↓CGG) are isoschizomers.

42. Compatible cohesive ends You may encounter situations where the sequence you are trying to clone does not have any appropriate restriction site available It is possible in some cases to find alternative restriction enzymes that cut sites that have different recognition sites but the same central four bases and produce the same overhangs, which can be ligated Example: EcoRI and MfeI EcoRI siteMfeI site

43. Tools for restriction site analysis of DNA sequences NEBcutter: –http://tools.neb.com/NEBcutter2/index.phphttp://tools.neb.com/NEBcutter2/index.php Webcutter: –http://rna.lundberg.gu.se/cutter2/http://rna.lundberg.gu.se/cutter2/

44. Ligation

45. DNA ligation DNA ligase - a DNA repair enzymes that is used to join free DNA ends together DNA ligase is dependent on energy (ATP) for the joining of DNA ends http://www.chups.jussieu.fr/polys/biochimie/BGbioch/POLY.Chp.3.3.html

46. DNA ligation T4 DNA ligase does not appear to have any role in nucleic acid metabolism in bacteriophage T4 infected E. coli, but instead appears to be required for the attachment of the bacteriophage’s tail fibers to its base plate during bacteriophage assembly T4 DNA ligase is available from several different suppliers (Fermentas, Roche, NEB, Epicentre…) Usually for molecular cloning DNA ligase from bacteriophage T4 is used (most versatile)

47. DNA ligation: Important principles Both blunt ends and “sticky” ends can be ligated with T4 DNA ligase Incubation temperature is a compromise between maximal enzymatic activity (the higher, the better – up to 37 o C) and stabilization of interactions of the DNA ends (the lower, the better – down to 4 o C)

48. Fast ligation kits Formerly, ligation was carried out overnight at 4 o C or for 4-6 hours at 15 o C Better enzyme preparations now allow for quicker ligation (room temp; 20-30 min for sticky ends, ~2 hrs for blunt ends) Nowadays, almost all manufacturers offer Fast ligation kits (5-15 min ligation time), e.g. –Epicentre Fast Link http://www.epibio.com/item.asp?ID=296 http://www.epibio.com/item.asp?ID=296 –NEB Quick ligation kit http://www.neb.com/nebecomm/products/productM2200.asp http://www.neb.com/nebecomm/products/productM2200.asp –Fermentas Rapid ligation kit http://www.fermentas.com/catalog/kits/kitrapidligation.htm http://www.fermentas.com/catalog/kits/kitrapidligation.htm All these kits probably use addition of polyethylene glycol (PEG) to improve ligation

49. A typical fast ligation reaction 50-200 ng Vector DNA Variable amount of insert DNA (1:1 – 1:3 Vector/Insert ratio for sticky ends; 1:5 for blunt ends) Ligation Buffer (Very low salt, pH7.5) ATP Ligase For Epicentre Fast Link (1:2 V/I ratio): 5.0 ul of 20 ng/ul Vector DNA ( 6.2kb) 2.0 ul of 30 ng/ul insert DNA (2.1 kb) 1.5 ul 10x buffer 1.5 ul of 10mM ATP 1.0 ul ligase 4.0 ul H 2 O 15.0 ul total Volume 5-15 minutes at room temperature (± 21 o C)

50. A useful ligation formula ng vector/ size (bp) vector = X/size (bp) insert Solve for X Multiply by 2 Example: –Vector is 6900 bp, insert is 750 bp –You decided to use 103 ng of vector –103ng/6900 bp = X/750 bp => 11.2 ng –x 2 = 22.4 ng (of insert) to get a 2:1 molar ratio of insert to vector

51. Reducing background: A) Cutting re-ligated plasmids To decrease background due to re-ligation of incompletely digested vector, the ligation reactions may be treated with a restriction enzyme that cuts within the re-ligated vector, but not the proper construct YFG (your favorite gene) KpnISalI  Inactivate ligase, adjust buffer, cut with SmaI, BamHI or XbaI, inactivate restriction enzyme  transform

52. B) Phosphatase Re-ligation can be prevented using Calf intestinal phosphatase (CIP) to remove terminal phosphates from the linearized vector prior to the ligation reaction – This may also require phosphorylation of the insert before the ligation reaction http://www.escience.ws/b572/L6/L6.htm

53. C) α-complementation Many laboratory E.coli strains allow for α- complementation for cloning –These strains are modified to express only the β-fragment (C- terminal) of β-galactosidase, which is inactive Wikipedia

54. C) α-complementation –The β-fragment of β-galactosidase needs interaction with the short α- fragment to form a functional enzyme –Many cloning vectors carry the α-fragment within the multiple cloning site Wikipedia

55. C) α-complementation –If the MCS of your vector is intact, transformed E.coli cells grown on IPTG will convert the compound X-gal to a blue dye, which turns the colonies blue, indicating that the vector re-ligated Wikipedia

56. Blue – white screen (α- complementation) to identify plasmid clones carrying inserts Wikipedia

57. General recommendations Keep a clean working environment Aliquot buffer, ATP Do not let ATP warm up to room temperature (the same is true for dNTPs in PCR) Heat inactivation of ligase (70 o C for 15-20 min) is recommended to reach optimal transformation frequencies

58. If you’re only interested in cloning a PCR product (and frame, etc. are not important) Cut vector with a blunt end cutter (e.g. SmaI or PvuII) Add purified PCR product at the proper concentration Ligate with blunt end method Transform E.coli

59. Plasmid preparation

60. Analysis of plasmid constructs Pick several transformant colonies, prepare DNA Perform analytical digests (10 ul digest volume may be enough) –First, digest with the enzyme(s) used to insert your DNA into the Vector –Second, digest candidates with a second (set of) enzyme(s) –For one of these digests, include uncut DNA as a control If any of the DNAs used in the construct were obtained by PCR: Sequence

61. Example: cloning of the human HIF-1  cDNA BamHIXbaI 2481

62. First digest: BamHI/ XbaI Expected sizes: 5.5 kb (pcDNA3) 2.5 kb (HIF-1  ) 5.5 kb 2.5 kb

63. BamHIXbaI SpeI 527 1954 Expected sizes 6 kb 1.9 kb Second Digest: BamHI/ SpeI

64. dam methylation Example: XbaI site is TCTAGA dam site is G m ATC (m= methylated base A) If you generate a XbaI site by PCR, but don’t pay attention to the following bases, you might end up with TCTAGATC The A in your restriction site will be methylated (TCTAG m ATC), and XbaI will not cut anymore once the DNA has passed through a dam+ E.coli strain You also have to verify restriction maps that indicate sites for enzymes that are sensitive to dam or dcm (or CpG) methylation. Be sure you check both ends!! (G m ATCTAGA is also methylated)

65. PCR

66. Generation of DNA fragments for cloning by PCR PCR = polymerase chain reaction Repeating cycles including: Denaturation (melting) of template DNA Annealing of an oligonucleotide primer Extension/elongation, that is new DNA synthesis by a (thermostable) DNA polymerase

67. Principle of PCR Exponential amplification of template DNA (25 cycles  [DNA at beginning] x 2 25  33 x10 6 fold amplification)

68. 1. Medical - Clinical diagnostic tests for some genetic disorders -Clinical diagnostic tests for various infectious agents -Blood screening to quantify the ammount of virus 2. Research - Molecular cloning - Genetic engineering - Mapping of the human genome 3. Forensic - Molecular archaeology and ancient DNA -Criminal investigations, parental testing etc. Applications of PCR

69. Requirements for PCR Thermocycler Thermostable DNA polymerase Template DNA (VERY little) Target-specific oligonucleotide primers Proper buffer conditions dNTPs

70. Thermocycler http://www.molecularstation.com/molecular-biology-images/509-pcr-pictures/71-thermocycler-old-pcr- machine.html?size=big

71. Thermostable DNA polymerases An automated thermocycler-based DNA amplification became possible only after Taq polymerase, that remains active even after DNA denaturation at 95 °C, was used. Purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C) environments such as hot springs of Yellowstone national park. Wikipedia

72. Thermostable DNA polymerases Available from a variety of sources –Taq (NEB and others), Dynazyme (Finnzymes): cheap, no proofreading activity; good for analytical PCR and short DNA fragments –Platinum Taq Hi Fi (Invitrogen), Dynazyme EXT (Finnzymes): proofreading activity, higher fidelity and longer products –Pfu (varying sources): proofreading activity, low processivity; good for high fidelity cloning of shorter DNA fragments –Pfu turbo (Stratagene): like Pfu, but better processivity; also good for longer PCR products (>4kb, up to over 10kb) –Pfu Ultra (Stratagene), Phusion (Finnzymes): Very high fidelity, good for long products –Many others….

73. Thermostable DNA polymerases http://www.kapabiosystems.com/products/name/kapa-hifi-pcr-kits Again, this is not an advertisement!!!! There are other very high fidelity polymerases too.

74. Concentrations of PCR components Vary depending on manufacturer and enzymes –  Check instructions carefully!! Typically: –10-250 ng of template DNA –Buffer with 1.5 mM MgCl 2 (final concentration) –200 uM of each dNTP –0.3-1.0 uM of each primer –Enzyme according to manufacturer

75. 1)Initialization. 95 °C (or 98 °C if extremely thermostable polymerase is used) for 1 min. 2)Denaturation. 95 °C for 50 seconds. Double-stranded DNA template is melted into two single-stranded DNA molecules. 3)Annealing. 50–65 °C for 50 seconds. The primers anneal to the single-stranded DNA template. Typically the annealing temperature is about 3-5 °C below the Tm (melting temperature) of the primers used. 4)Extension/elongation. Temperature depends on polymerase used (72 °C for Taq). A new DNA strand complementary to the DNA template is synthesized. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified (about thousand bases per minute). 5)Final elongation. 72 °C for 5–15 min. 6)Final hold. 4°C for an indefinite time for short-term storage of the reaction products. PCR conditions 25-35 cycles

76. PCR conditions Manufacturer’s instructions – Finnzymes Dynazyme/Dynazyme EXT: http://www.finnzymes.com/pcr/pcr-tech_appendix.html http://www.finnzymes.com/instruction_manuals.html http://www.finnzymes.com/pcr/pcr-tech_appendix.html http://www.finnzymes.com/instruction_manuals.html –Finnzymes Phusion: http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html –Taq polymerase http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0273.pdf http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0273.pdf –Pfu http://www.stratagene.com/manuals/600135.pdf http://www.stratagene.com/manuals/600135.pdf –Pfu turbo http://www.stratagene.com/manuals/600250.pdf http://www.stratagene.com/manuals/600250.pdf

77. Things to pay attention to Avoid the use of buffers containing EDTA –Because of the low concentration of MgCl 2, PCR reactions are sensitive even to small amounts of EDTA Always assemble PCR reactions on ice –There is a danger of getting unspecific PCR products due to low enzymatic activity at room temperature –If you encounter problems with unspecific products, try a Hot Start (adding the enzyme just before the first elongation cycle) or use a Hot Start enzyme preparation

78. Things to pay attention to Prepare PCR in a clean working environment

79. PCR sensitivity Sensitivity is both a blessing and a curse for people who use it to analyze DNA. PCR is very vulnerable to contamination (like DNA samples from investigator’s own body) The ability to amplify everything in the sample means that a PCR can be used to find DNA which may only be present in trace amounts in a sample (perhaps as low as 1 molecule).

80. PCR sensitivity Neanderthal genomic DNA was sequenced using material obtained from fossilized bones that are tens of thousands of years old (despite contaminations with microbal and modern human’s DNA).

81. PCR sensitivity Neanderthal genomic DNA was sequenced using material obtained from fossilized bones that are tens of thousands of years old (despite contaminations with microbal and modern human’s DNA). http://ideonexus.com/2012/04/16/adventures-in-personal-genomics/

82. Primer design For a regular PCR reaction, you need two primers, annealing to your template –The 5’ primer has a sequence identical to a sequence in the upper (“sense” if it is a protein coding sequence) strand –The 3’ primer has a sequence that is the Reverse complement of the sequence on the upper strand 5’-GATTCATATTCCGATGACTTGAGCTTGGGAATTCGTAGCTATGCAGAATGCTG-3’ 5 ’-X…XGATTCATATTCCGATGACTTG- 3 ’ 5’-primer 3 ’-GCATCGATACGTCTTACGACX..X- 5 ’ 3’-primer 5’- primer: 5’-X…XGATTCATATTCCGATGACTTG-3’ 3’- primer: 5’-X…XCAGCATTCTGCATAGCTACG-3’

83. Primer design Length (depending on template source) 18-25 bp homologous to gene/cDNA intended for cloning The last (3’-most) base should be a G or a C Addition of required restriction site + extra bases 5’ to restriction site (3-6?) Kozak sequence CCACC before ATG ? If you have a choice – 50% GC content –Avoid long runs (>3) of any one nucleotide –Avoid primer dimers and strong secondary structure

84. Tools for primer design Reverse complement –Changes any nucleotide sequence into its properly oriented reverse complement –http://www.bioinformatics.org/sms/rev_comp.htmlhttp://www.bioinformatics.org/sms/rev_comp.html DNA/oligonucleotide calculator –Calculates melting temperature, secondary structure, primer dimer formation etc., e.g. –http://www.sigma-genosys.com/calc/DNACalc.asphttp://www.sigma-genosys.com/calc/DNACalc.asp –http://www.thermoscientificbio.com/webtools/tmc/

85. Food for thought: What REALLY is the melting temperature of your oligonucleotide?? Very often, only part of your primers is actually annealing to the template! Restriction sites and extra 5’ bases rarely share any homology with the template DNA Melting temperature is calculated based on various formulas like Tm = 2°C(A+T) + 4°C(G+C) [for 14-20 bp oligos at 0.9M NaCl] Tm = 81.5 + 16.6 log M + 41(XG+XC) - 500/L - 0.62F [for oligos > 50 bp] M is the molar concentration of monovalent cations, XG and XC are the mole fractions of G and C in the oligo, L is the length of the shortest strand in the duplex, and F is the molar concentration of formamide.

86. An example: 5’…..CTAGGCCTTAGATTCATATTCCGATGACTTGAGCTTGGGAATTCGTAGCTATGCAGAATGCTG….-3 ’ 5’-TTATCTCGAGGATTCATATTCCGATGACTTG-3’ XhoI site In rare cases, you may not get any product at all, or get unspecific product…

87. Is there a solution? Most PCRs appear to work well under “Standard” conditions (annealing temperature of 54-56 o C) If you have problems getting any product, you may –Try a PCR reaction with an annealing temperature adjusted to the “homologous” part of your primers (you can also run the first ten or so cycles at this annealing temperature and then increase it) –Determine the optimal annealing temperature using a gradient PCR machine (reaction will be performed at different annealing temperatures, with all other values being the same).

88. Processing and verification of PCR product If the product was obtained by PCR from genomic DNA: –Run out a small aliquot (e.g. 3-5 ul) on gel to determine if product is present and has right size –If O.K.  purify the rest of the product directly from PCR mix If product was obtained from a plasmid template –Run out entire reaction on agarose gel, check size and purify from gel Ultimate verification: always sequence PCR products!

89. Materials/assumed conditions for assignment Enzymes: –PCR: Finnzymes Phusion protocol –Site-directed mutagenesis: Stratagene protocol –Restriction digests: NEB enzymes https://www.neb.com/tools-and- resources/interactive-tools/double-digest-finder –Ligation: Epicentre Fast-Link™ DNA Ligation Kit

90. Information to be included on your assignment solutions Restriction Maps of vector and insert PCR and Mutagenesis: –Oligonucleotide sequences –PCR reaction mix –Cycling conditions –Processing? (Analytical gel + direct purification or preparative agarose gel + how can you be sure that the mutagenesis works?) Preparative restriction digests: –Digest mixes  indicate enzymes and units used, indicate DNA concentrations Ligation: –Ligation mix Analytical restriction digest –Digest mixes –Expected restriction patterns for the first and second digest (you may also want to draw a circular map)

91. Useful Databases for assignments PubMed (Nucleotide) –http://www.ncbi.nlm.nih.gov/nucleotidehttp://www.ncbi.nlm.nih.gov/nucleotide Stratagene (Site-directed mutagenesis) –http://www.tufts.edu/~mcourt01/Documents/Stratagene%20Quik change%20mutagenesis.pdfhttp://www.tufts.edu/~mcourt01/Documents/Stratagene%20Quik change%20mutagenesis.pdf (NOTE: only page number five is needed for the primer design - not the entire manual) Invitrogen –http://www.invitrogen.com/site/us/en/home.htmlhttp://www.invitrogen.com/site/us/en/home.html Expert Protein Analysis System –http://au.expasy.org/http://au.expasy.org/ Human Protein Reference Database –http://www.hprd.org/http://www.hprd.org/

92. Vector databases http://www.addgene.org/pgvec1 Lablife http://www.addgene.org/pgvec1?vectorid=4637&f=d&cm d=showvecinfohttp://www.addgene.org/pgvec1?vectorid=4637&f=d&cm d=showvecinfo http://www.neb.com/nebecomm/tech_reference/restrictio n_enzymes/dna_sequences_maps.asphttp://www.neb.com/nebecomm/tech_reference/restrictio n_enzymes/dna_sequences_maps.asp http://www.fermentas.com/techinfo/nucleicacids/mappblu escriptiiskks.htmhttp://www.fermentas.com/techinfo/nucleicacids/mappblu escriptiiskks.htm

93. Deadline and submission Assignments have to be sent in by If possible not larger than 1 Mb Please send to: Anatoliy.Samoylenko@oulu.fi