Molecular Biology II

Molecular cloning and mouse knockout principles

1- Molecular cloning

The tools that fuel molecular cloning

• Restriction digest of DNA• DNA ligation• PCR

https://www.neb.com/products/restriction-endonucleases

The Nobel Prize in Physiology or Medicine 1978jointly 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.

The tools that fuel molecular cloning

• Restriction digest of DNA• DNA ligation• PCR

https://www.neb.com/products/restriction-endonucleases

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

Restriction Endonucleases

• 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 TGAN8TGCT)

•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

Sites of methylation in bacteria, recognized by restriction endonucleases

http://www.mikeblaber.org/oldwine

Restriction Endonucleases

• 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.

• 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.

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

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/biochemistry/halford/research.html

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

Type II Restriction Endonucleases

Wikipedia

Crystal structure of EcoRI bound to DNA

Frequency of restriction sites

• If the base composition is assumed to be completely random, the restriction site frequency can be calculated as 4x, with X being the length of the recognition sequence

For a four-base cutter: 44 = 256 one cutting site every 256 bp

“ “ six “ “ : 46 = 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.

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

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

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?

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 recombinantsourse

Can digest 1 µg DNA in 5 min

Passed the blue-white selection

Heat inactivation possible

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.

• The unit definition refers to cleavage of linear (phage λ) DNA

• But: Plasmid DNA is usually supercoiled! *

*More about this later!

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

Source of enzyme

Source of enzymeNocardia (yellow)

Wikipedia

American Type Culture Collection

Application Oxidizes alkylbenzenesProduces restriction endonuclease NcoI

Isolation Soil

Type Strain no

Biosafety Level 1

Product Format freeze-dried

Price €427.00

Reaction buffer features

1X NEBuffer 3:100 mM NaCl50 mM Tris-HCl10 mM MgCl21 mM Dithiothreitol

pH 7.9 @ 25°C

1X NEBuffer 2:50 mM NaCl10 mM Tris-HCl10 mM MgCl21 mM Dithiothreitol

pH 7.9 @ 25°C

1X NEBuffer 4:50 mM potassium acetate20 mM Tris-acetate10 mM Magnesium Acetate1 mM DithiothreitolpH 7.9 @ 25°C

1X NEBuffer 1:10 mM Bis-Tris-Propane-HCl10 mM MgCl21 mM DithiothreitolpH 7.0 @ 25°C

Very low salt,

Low pH

Low salt High salt Low salt,

Acetate buffer

Buffer compatibility

• Some enzymes require special buffers• Sometimes additives are required BSA = bovine serum albuminSAM = 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

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!

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

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)

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

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).

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

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 methylationhttp://clincancerres.aacrjournals.org/content/11/18/6409/F1.expansion

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)

Supercoiledform

Relaxedform

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

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

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)

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.

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 site MfeI site

Tools for restriction site analysis of DNA sequences

• NEBcutter:

– http://tools.neb.com/NEBcutter2/index.php

• Webcutter:

– http://rna.lundberg.gu.se/cutter2/

Ligation

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

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)

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 37oC) and stabilization of interactions of the DNA ends (the lower, the better – down to 4oC)

Fast ligation kits• Formerly, ligation was carried out overnight at 4oC or for

4-6 hours at 15oC• 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

– NEB Quick ligation kit http://www.neb.com/nebecomm/products/productM2200.asp

– Fermentas Rapid ligation kit http://www.fermentas.com/catalog/kits/kitrapidligation.htm

• All these kits probably use addition of polyethylene glycol (PEG) to improve ligation

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 H2O

15.0 ul total Volume

5-15 minutes at room temperature (± 21oC)

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

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)

KpnI SalI

Inactivate ligase, adjust buffer, cut with SmaI, BamHI or XbaI, inactivate restriction enzyme transform

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

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

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

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

Blue – white screen (α-complementation) to identify plasmid

clones carrying inserts

Wikipedia

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 (70oC for 15-20 min) is recommended to reach optimal transformation frequencies

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

Plasmid preparation

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

Example: cloning of the human HIF-1 cDNA

BamHI XbaI

2481

First digest: BamHI/ XbaI

Expected sizes:

5.5 kb (pcDNA3)

2.5 kb (HIF-1)

5.5 kb

2.5 kb

BamHI XbaI

SpeI

5271954

Expected sizes

6 kb

1.9 kb

Second Digest: BamHI/ SpeI

dam methylation• Example: XbaI site is TCTAGA• dam site is GmATC (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 (TCTAGmATC), 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!! (GmATCTAGA is also methylated)

PCR

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

Principle of PCR

Exponential amplification of template DNA (25 cycles [DNA at beginning] x 225 33 x106 fold amplification)

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

Requirements for PCR

• Thermocycler

• Thermostable DNA polymerase

• Template DNA (VERY little)

• Target-specific oligonucleotide primers

• Proper buffer conditions

• dNTPs

Thermocycler

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

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

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….

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.

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 MgCl2 (final concentration)– 200 uM of each dNTP– 0.3-1.0 uM of each primer– Enzyme according to manufacturer

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

25-35cycles

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

– Finnzymes Phusion: http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html

– Taq polymerase http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0273.pdf

– Pfu http://www.stratagene.com/manuals/600135.pdf

– Pfu turbo http://www.stratagene.com/manuals/600250.pdf

Things to pay attention to

• Avoid the use of buffers containing EDTA– Because of the low concentration of MgCl2, 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

Things to pay attention to• Prepare PCR in a clean working environment

PCR sensitivitySensitivity 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).

PCR sensitivityNeanderthal 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).

PCR sensitivityNeanderthal 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/

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’

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

Tools for primer design

• Reverse complement– Changes any nucleotide sequence into its

properly oriented reverse complement– http://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.asp– http://www.thermoscientificbio.com/webtools/tmc/

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.

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…

Is there a solution?• Most PCRs appear to work well under “Standard”

conditions (annealing temperature of 54-56oC)• 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).

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!

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

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)

Useful Databases for assignments

• PubMed (Nucleotide)– http://www.ncbi.nlm.nih.gov/nucleotide

• Stratagene (Site-directed mutagenesis)– http://www.tufts.edu/~mcourt01/Documents/Stratagene

%20Quikchange%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.html

• Expert Protein Analysis System– http://au.expasy.org/

• Human Protein Reference Database– http://www.hprd.org/

Vector databases

• http://www.addgene.org/pgvec1• Lablife• http://www.addgene.org/pgvec1?

vectorid=4637&f=d&cmd=showvecinfo• http://www.neb.com/nebecomm/tech_reference/

restriction_enzymes/dna_sequences_maps.asp• http://www.fermentas.com/techinfo/nucleicacids/

mappbluescriptiiskks.htm

Deadline and submission

• Assignments have to be sent in by

• If possible not larger than 1 Mb

• Please send to: Anatoliy.Samoylenko@oulu.fi