Graduate lab manual
a practical guide
September 2008
Christianna Choulaki
INDEX
Glossary
Genomic DNA isolation by disruption method
Remarks
Methods
1) from peripheral blood leukocytes
2) frommousetails
PCR
Procedure
To minimize contamination risk
Agarose gel analysis of PCR products
Practical modifications to the PCR technique
Endonuclease digestion
Fragment complementarity and splicing
Restriction enzymes as tools
General protocol
hands-on
PBMC’s isolation with Ficoll (Histopaque-1077, Sigma)
Cell counting in Neubauer chamber
FACS
Principle
Flow cytometers
Fluorescent labels
Measurable parameters
Applications
hands on
Surface staining
FACS ANALYSIS-ELITE CYTOMETER
Total RNA isolation
A) Using Tri reagent (Sigma, cat# T 9424)
B) Using columns
Determine concentration
cDNA preparation (Reverse Transcription, RT)
Western Blot......
Tissue preparation
Gel electrophoresis
Transfer
Blocking
Detection
Analysis......
Colorimetric detection......
Chemiluminescence......
Secondary probing
Glossary
APSAmmonium PerSulfate
DTTDi ThioTheitol
ddH2Odouble distilled Water
dNTPsdeoxyNucleoTides Phosphate
EtOHEthanol
FCSFetal Bovine Serum
FicollHistopaque-1077, Sigma
gDNAgenomic DNA
HBSSHank’s Balanced Salt Solution
KOKnock Out
PBMCsPeripheral Blood Mononuclear Cells
PBSPhosphate Buffer Saline
qPCRQuantitative PCR (ral time PCR)
RTRoom Temperature
RT-PCRReverse Transcription PCR
SDSS Dodecyl Sulfate
TBETris Borate EDTA buffer
TBSTris Buffer Saline
TBS-TTris Buffer Saline Tween
TETis EDTA
TEMEDN,N,N,N’,N’-teteramethylethylenediamine
Trishydroxymethyl aminomethane
TSETris Saline EDTA
UVultraviolet
WTWild Type
W/Vweight per volume
Genomic DNA isolation by disruption method
Remarks
Eukaryotic DNA is a double stranded, relatively stable molecule. However, introduction of nucleases to DNA solutions should be avoided as these enzymes will degrade DNA. Genomic DNA consists of very large DNA molecules, which are fragile and can break easily. To ensure the integrity of gDNA, excessive and rough pipeting and vortexing should be avoided. DNA is subject to acid hydrolysis when stored in water, and should therefore be stored in TE buffer.Complete disruption and lysis of cell walls and plasma membranes of cells and organelles is an absolute requirement for all genomic DNA isolation procedures. Incomplete disruption results in significantly reduced yields
Disruption method is based on the fact that the lysis buffer containsSDS, a detergent that breaks down cellular membranes and proteinase K, a protease that digests protein cellular components, resulting in the liberation of nucleic acids in the solution.
RNase is degrading RNA without interfering with DNA. Phenol/chloroform treatment sequestrates proteins in the interphase, while chloroform absorbs phenol remainings from the aqueous phase. Isopropanol adsorbs water molecules, resulting in a “concentration”of DNA molecules witch facilitates their precipitation. The ionic strength of the buffer is also very important as it neutralizes negative charges of the DNA molecules. Ethanol 70% wash replaces isopropanol molecules (which can inhibit subsequent enzymatic processes) with the more volatile Ethanol, rehydrates DNA making the pellet easier to redissolve and removes co-precipitated salts.
Methods
1) from peripheral blood leukocytes
5ml peripheral blood in 50 ml tube containing 200μl EDTA0.5M(to prevent coagulation)
spin at 4000rpm for 15min at 4°C.
Remove serum and collect the white phase (leucocytes+reticulocytes)
cell disruption
+ an equal volume of TSE bufferNaCl 150mM
EDTA pH8 100mM
Tris pH8 20 mM
+ SDS at a final concentration of 1%w/v
+ proteinase K at a final concentration of 10μg/ml
incubate 65°C 45-60 min
RNaseA treatment
+RNAseI at a final concentration of 100μg/ml
incubate at 37°C for 30min
DNA extraction
+ an equal volume of phenol:chloroform (1:1)
mix gently for 5 min
spin at 4000rpm for 15 min at RT
carefully aspirate aqueous phase (containing nucleic acids) without taking any proteins from the interphase and put in a new tube
repeat the phenol extraction till the interphase becomes clear
repeat the extraction with chlorophorm only (to absorb phenol remnants)
DNA precipitation
+ 1/10 volumes NaAcetate3M, pH5.4 and mix
+ 2,2 volumes Ethanol -20°C
gently mix, watch the DNA filament formation
spin it around a flame-rounded Pasteur pipette
wash in 70% ethanol -20°C (desalting)
leave on air for some minutes to evaporate ethanol
dissolve DNA by immersion of the edge of the Pasteur pipette in a 1.7ml tube containing 0.5 ml TE
Determine concentration by photometry:
For double stranded DNA, a solution containing 50μg/ml produce an OD260nm =1 (in conventional photometers)
OD260nm =1 C=50μg/ml gDNA
hands on
(for a single beem photometer)
Dilution:add 5μλ DNA suspension in0.995 ml dd H20 (dilution factor=200)
insert quartz cuvette in the photometer, fill it with ddH20
Set BLANK
remove ddH20 from the cuvette, fill it with the sample
note the OD260nm, and the OD280nm
the concentration of the sample is given by the formula
C=OD260nmx dilution factor x 50μg/ml
2) frommousetails
Put a piece of 5 mm mouse tail in eppendorf tube
Add 700μl proteinase K bufferTris pH8 50 mM
EDTA pH8 100mM
NaCl 100mM
SDS 1%
+ proteinase K at a final concentration of 360 μg/ml, mix well
o/n 55°C
Add RNase I at a final concentration of 140 μg/ml
37°C 1-2hours
Add 700μl phenol/chloroform (1:1), shake 15min (NO VORTEX)
Spin 15min, RT, 13.000rpm
Collect aqueous (upper) phase in a fresh tube
Add 700μl chloroform
Shake 15 min
Spin 15min, RT, 13.000rpm
Collect aqueous (upper) phase in a fresh tube
Add 700μl isopropanol
Gentle mix
If a visible pellet is formed, fish it out with a sealed Pasteur pipette
If pellet not visible, spin 15min, RT, 13.000rpm, decant supernatant.
Pellet might have a glassy appearance, care should be taken when removing the supernatant as isopropanol precipitation pellets are more loosely attached to the side of the tube.
Wash with 1 ml EtOH70%, brief vortex, spin 5min at 4°C, decant supernatant, absorb residual liquid with a pipette tip
Take care not to dislodge DNA pellet
Air dry 10-15min (avoid over drying as this make DNA difficult to redisolve
Resuspend in appropriate V of H2O or TE
Let stand 15 min at RT, o/n at 4°C or, if difficult to ressuspend, o/n at RT or even at 55°C for 1-2 hours.
Verify that DNA is dissolved
Determine concentration by spectrophotometry and keep at -20°C
Determine concentration by photometry:
Add 5μl DNA suspension in 995μl H20 (dilution factor=200)
for OD260nm =1 C=50μg/ml gDNA
the concentration of the sample is given by the formula:
C= OD260nm* dilution factor * 50μg/ml
PCR
PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or a non-coding sequence.
PCR, as currently practiced, requires several basic components:
- DNA templatethat contains the region of the DNA fragment to be amplified
- primers, which are complementary to the DNA regions at the 5' and 3' ends of the DNA region that is to be amplified.
- a DNA polymerase (e.g. Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C), used to synthesize a DNA copy of the region to be amplified
- Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
- Buffer solution, which provides a suitable chemical environment for optimum activity and stability of the DNA polymerase
- Divalentcation. generally Mg2+ is used (Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis)
- Monovalent cation(potassium ions)
For atypical 20μl PCR reaction
TemplateGenomic DNA ≈100ng
or
cloned DNA ≈50ng
or
cDNA ≈1-2μl of the reverse transcription reaction
Polymerase buffer1X
MgCl21,5-2,5 mM
dNTPs200 μM
Primers0,25-0,5 μM each
Polymerase0,3-0.5 units
Always keep in mind that PCR is a very sensitive technique
The PCR is carried out in small reaction tubes (0.2-0.5 ml volume), containing a reaction volume typically of 15-100 μl, that are inserted into a thermal cycler. This is an instrument that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. Most thermal cyclers have heated lids to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation.
Procedure
The PCR usually consists of a series of 20 to 35 cycles. Most commonly, PCR is carried out in three steps, often preceded by one temperature hold at the start and followed by one hold at the end.
- Prior to the first cycle, during an initialization step, the PCR reaction is often heated to a temperature of 94-96°C, and this temperature is then held for 1-10 minutes. This first hold is employed to ensure that most of the DNA template and primers are denatured, i.e., that the DNA is melted by disrupting the hydrogen bonds between complementary bases of the DNA strands. (Also, some PCRs require this step for activation ofhot-start polymerase). Following this hold, cycling begins, with one step at 94-98°C for 20-30 seconds (denaturation step).
- The denaturation is followed by the annealing step. In this step the reaction temperature is lowered so that the primers can anneal to the single-stranded DNA template. Brownian motion causes the primers to move around, and DNA-DNA hydrogen bonds are constantly formed and broken between primer and template. Stable bonds are only formed when the primer sequence exactly matches the template sequence, and to this short section of double-stranded DNA the polymerase attaches and begins DNA synthesis. The temperature at this step depends on the melting temperature of the primers, and is usually between 50-64°C for 20-40 seconds.
- The annealing step is followed by an extension/elongation step during which the DNA polymerase synthesizes new DNA strands complementary to the DNA template strands. The temperature at this step depends on the DNA polymerase used. Taq polymerase has a temperature optimum of 70-74°C; thus, in most cases a temperature of 72°C is used. The hydrogen bonds between the extended primer and the DNA template are now strong enough to withstand forces breaking these attractions at the higher temperature. Primers that have annealed to DNA regions with mismatching bases dissociate from the template and are not extended. The polymerase adds dNTP's that are complementary to the template in 5' to 3' direction, thus reading the template in 3' to 5' direction. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases in one minute. A final elongation step of 5-15 minutes (depending on the length of the DNA template) after the last cycle may be used to ensure that any remaining single-stranded DNA is fully extended. A final hold of 4-15°C for an indefinite time may be employed for short-term storage of the reaction, e.g., if reactions are run overnight.
To minimize contamination risk
work in a template free environment
clean you bench periodically with 10% chlorine
keep your pre-PCR reagent tubes capped as long as possible
prepare reaction mixes and always add template last
To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as amplimer), agarose gel electrophoresis is commonly employed for size separation of the PCR products. The size(s) of PCR products is thereby determined by comparison with a DNA ladder, which contains DNA fragments of known size, ran on the gel alongside the PCR products.
the agarose gel is a matrix the density of which is determined by the concentration of agarose. Single stranded DNA is a negatively charged molecule that migrates through the gel towards the +pole of the field (cathode) at a rate inversely proportional to its size. That means that larger molecules migrate more slowly. Ethidium Bromideis a fluorescent dye that intercalates between the bases. DNA bands are visualised upon excitation with UV light.
Agarose gel analysis of PCR products
1
Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify the IGF gene from3 different DNA samples. In sample #1 the gene was not amplified by PCR, whereas bands for tissue #2 and #3 indicate successful amplification. A positive control, and a DNA ladder containing DNA fragments of defined length (last lane to the right) to estimate fragment sizes in the experimental PCRs, were also ran.
1
Practical modifications to the PCR technique
- RT-PCR - RT-PCR (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissueRNA. The PCR reaction is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene.
- Quantitative PCR - Q-PCR (Quantitative PCR) is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see above), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
- Hot-start PCR is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by simply heating the reaction components briefly at the melting temperature (e.g., 95˚C) before adding the polymerase.Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
- Multiplex-PCR - The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
Endonuclease digestion
A restriction enzyme (or restriction endonuclease) is an enzymethat cuts double-stranded DNA. The enzymemakes two incisions, one through each of the sugar-phosphate backbones (i.e., each strand) of the double helix without damaging the nitrogenous bases.
The chemical bonds that the enzymes cleave can be reformed by other enzymes known as ligases, so that restriction fragments carved from different chromosomes or genes can be spliced together, provided their ends are complementary (more below). Many of the procedures of molecular biology and genetic engineering rely on restriction enzymes. The term restriction comes from the fact that these enzymes were discovered in E. coli strains that appeared to be restricting the infection by certain bacteriophages. Restriction enzymes therefore are believed to be a mechanism evolved by bacteria to resist viral attack and to help in the removal of viral sequences. They are part of what is called the restriction modification system.
Fragment complementarity and splicing
EcoRI cleavage produces "sticky" ends
SmaI restriction enzyme cleavage produces "blunt" ends
Because recognition sequences and cleavage sites differ between restriction enzymes, the length and the exact sequence of a sticky-end "overhang", as well as whether it is the 5' end or the 3' end strand that overhangs, depends on which enzyme produced it. Base-pairing between overhangs with complementary sequences enables two fragments to be joined or "spliced" by a DNA ligase. A sticky-end fragment can be ligated not only to the fragment from which it was originally cleaved, but also to any other fragment with a compatible sticky end.The sticky end is also called a cohesive end or complementry end in some reference. If a restriction enzyme has a non-degenerate palindromic cleavage site, all ends that it produces are compatible. Ends produced by different enzymes may also be compatible. Knowledge of cleavage sites allows molecular biologists to anticipate which fragments can be joined in which ways, and to choose enzymes appropriately.
Restriction enzymes as tools
Recognition sequences typically are only four to twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides--A,C,G and T--into a four or eight or twelve nucleotide sequence, recognition sequences tend to "crop up" by chance in any long sequence. Furthermore, restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories. As a result, potential "restriction sites" appear in almost any gene or chromosome. Meanwhile, the sequences of some artificial plasmids include a "linker" that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. So no matter the context in which a gene naturally appears, there is probably a pair of restriction enzymes that can snip it out, and which will produce ends that enable the gene to be spliced into a "plasmid" (i.e. which will enable what molecular biologists call "cloning" of the gene).
Another use of restriction enzymes can be to find specific SNPs. If a restriction enzyme can be found such that it cuts only one possible allele of a section of DNA (that is, the alternate nucleotide of the SNP causes the restriction site to no longer exist within the section of DNA), this restriction enzyme can be used to genotype the sample without completely sequencing it. The sample is first run in a restriction digest to cut the DNA, and thengel electrophoresis is performed on this digest. If the sample is homozygous for the common allele, the result will be two bands of DNA, because the cut will have occurred at the restriction site. If the sample is homozygous for the rarer allele, the sample will show only one band, because it will not have been cut. If the sample is heterozygous at that SNP, there will be three bands of DNA.