General PCR Protocols and Its Product Processes
Recommended Reagent Concentrations
Recommended Reaction Conditions
Initial Conditions
Temperature Cycling
"Hot Start" PCR
Asymmetric PCR for ssDNA Production
Detecting Products
Labelling PCR Products with Digoxigenin
Cleaning PCR Products
Sequencing PCR Products
Cloning PCR Products
AND ALWAYS REMEMBER:
Protocol for PCR using Taq DNA Polymerase
Protocol for PCR with Taq DNA Polymerase. Avoiding Contamination. PCR allows the production of more than 10 million copies of a target DNA sequence from ...www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm -
General PCR Protocol
Detailed PCR protocol from the web site of the Department of Biology, University of Michigan, USA.www.mcdb.lsa.umich.edu/labs/maddock/protocols/PCR/general_pcr_protocol.html
Standard PCR
However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. ...www.mcb.uct.ac.za/pcrcond.htm
PCR PROTOCOL
PCR PROTOCOL FOR cDNA ARRAYS ON MEMBRANES. Purpose: to amplify insert DNA from purified plasmid DNA derived from bacterial. plasmid libraries. ...www.daf.jhmi.edu/microarray/protocols/protocol6.pdf
Basic PCR Protocol
Basic PCR Protocol. CGLab, 7/2002. 1). Wipe down the bench area with bleach and a new paper towel. 2). Take the PCR components out of the freezer to thaw ...www.sfsu.edu/~biology/cgl/media/PCR%20Protocol-Basic.pdf
Long PCR Protocol
Protocol and guidelines for choice of conditions for PCR of long sequences (10 kb or larger). From Genetics Dept., Harvard Medical School,Boston, MA, USA.arep.med.harvard.edu/labgc/estep/longPCR_protocol.html
20-mer Polymerase Chain Reaction Procedure (for MJ Research ...
MJ Research thermal Cycler: 10-mer PCR for amplification of random genomic DNA fragments ... Edit (or choose a program if it has been set up) PCR Program. ...wheat.pw.usda.gov/~lazo/methods/lazo/pcrproto.html
Single tube confirmation PCR protocol
For characterization colonies of transformed clones of Saccharaomyces, from the web site of the Stanford Genome Technology Center, Palo Alto, CA, USA.www-sequence.stanford.edu/group/yeast_deletion_project/single_tube_protocol.html
Protocol for PCR with Hot Start Taq DNA Polymerase
Protocol for PCR with Hot Start Taq DNA Polymerase. How to Avoid Contamination. During PCR, usually more than 10 million copies of a template DNA can be ...www.fermentas.com/profiles/modifyingenzymes/pdf/protocols/protocolhotstart.pdf
A Basic Polymerase Chain Reaction Protocol
Here, a basic, straight-forward PCR protocol is. presented. Where appropriate, some of the choices for modifying this standard reaction ...www.idtdna.com/support/technical/TechnicalBulletinPDF/A_Basic_PCR_Protocol.pdf
PCR Reamplification Protocol
PCR Reamplification for Inadequate or Failed Amplifications. Change your standard PCR protocol for the locus as follows:. decrease the number of cycles by ...genome-lab.ucdavis.edu/Protocols/pcr_tips/pcr_reamplification.htm
Inverse PCR and Sequencing Protocol
Inverse PCR and Sequencing Protocol on 5 Fly Preps. For recovery of sequences flanking XP elements. This protocol is an adaptation of ...flystocks.bio.indiana.edu/pdfs/Exel_links/5__fly_iPCR_XP_pub.pdf
Saturday, September 13, 2008
Genotyping by PCR
Methods for Mouse Genotyping by PCR (protocol 1)
1. Preparation of genomic DNA from the mouse tail.
1) Obtain about 5 mm of the mouse tail and cut it symmetrically into two pieces.
Note: Too long tail can result in the inhibition of PCR because of increased impurity.
Put the cut tail into 500 ul lysis buffer 9see below) in a 1.5 ml microfuge tube, which should be
with a rubber ring to prevent leakage of the content. Without DNA degration, tails can be stored at
-80 centigrade even after standing at room temperature for a couple of hours.
2) Incubate at 65 degree centigrade with gentle shaking overnight. When a part of tail tissue remains
because of inactivation of Proteinase K by the high temperature, addition of more Proteinase K is
recommended to lyse the tail completely.
3)--This step is optional--
Detect the quality of the genomic DNA by 1.0% agarose gel electrophoresis. 10 ul of the lysate is
enough for the detection. The sample may not be suitable for the following PCR unless >4kb DNA
is detected.
4) Heat the lysates at 95 degree centigrade for 10 minutes in a PCR machine or by boiling to inactivate
Proteinase K completely.
5) Spin the tail lysate briefly before transferring to a PCR tube to exclude the tissue debris. Proceed
directly to PCR using the tail DNA lysate as a template at a volume rate of 1/10 as follows.
2. PCR reactions.
Contents of PCR mixture for wildtype/knockout allele screening:
5 ul tail DNA solution: spin briefly before transferring to a PCR tube to avoid contamination of debris.
1 ul 10 uM primers (each upper and lower primer)
5 ul 10x KOD dash DNA polymerase (from TOYOBO Co. LTD.,Japan)
5 ul 2.0 mM dNTPs
32 ul dd H2O
Total volume of 50 ul
We recently found that the final volume can be reduced to 25 ul without mineral oil application.
Sequences of PCR primers: should be designed according to your target gene.
Primers for detecting wild-type allele
Primers for detecting knock-out allele
Methods for Mouse Genotyping by PCR (protocol 2)
Transgenic Genotyping from Tail Biopsies
Harvard University--MCB Department / HSCI
Remove .5-1 cm of the tail and place in 1.5 ml Eppendorf tube. (Store at -20oC until ready to digest).
Digest in Lysis Buffer* + Proteinase K (to 200 ug/ml final conc.).
Incubate in 55oC water bath overnight. (Vortex 1x after 1-2 h).
Add .5 ml Phenol:Chloroform:Isoamyl alcohol (25:24:1) to each tube and vortex for 30 sec.
Spin at top speed in a microcentrifuge for 5 minutes.
Transfer upper (aqueous) phase to new tube; make sure no debris from the interface is transferred.
Add 1 ml of 100% EtOH.
Vortex briefly or shake. Stringy white precipitate (the genomic DNA) should now be visible.
Spin briefly (<1 min) just enough to get the DNA to cling to the plastic, and decant supernatant.
Wash with 1 ml of 70% EtOH.
Let air dry until the pellet becomes partially translucent, but do NOT over-dry, or the DNA will not go into solution any longer.
Redissolve the pellet in 100 ml TE, pH 8.0.
Check concentration, and calculate the total yield, which should be around 10 to 50 mg.
Use 100 ng for subsequent PCR analysis.
*Lysis Buffer:
10 mM Tris-HCl, pH 8.0
25 mM EDTA, pH 8.0
100 mM NaCl
0.5% SDS
DNA from Tail Biopsies
Genotyping Transgenic Rodents by PCR
Isolation of DNA from Mouse Tail Biopsies
Lac-Z Detection in Tail Biopsies
Preparation of Mouse Tail DNA for Dot Blots or PCR
Universal Mouse Genotyping Protocol Using PCR
beta globin Primers
lacZ Primers
neo Primers
1. Preparation of genomic DNA from the mouse tail.
1) Obtain about 5 mm of the mouse tail and cut it symmetrically into two pieces.
Note: Too long tail can result in the inhibition of PCR because of increased impurity.
Put the cut tail into 500 ul lysis buffer 9see below) in a 1.5 ml microfuge tube, which should be
with a rubber ring to prevent leakage of the content. Without DNA degration, tails can be stored at
-80 centigrade even after standing at room temperature for a couple of hours.
2) Incubate at 65 degree centigrade with gentle shaking overnight. When a part of tail tissue remains
because of inactivation of Proteinase K by the high temperature, addition of more Proteinase K is
recommended to lyse the tail completely.
3)--This step is optional--
Detect the quality of the genomic DNA by 1.0% agarose gel electrophoresis. 10 ul of the lysate is
enough for the detection. The sample may not be suitable for the following PCR unless >4kb DNA
is detected.
4) Heat the lysates at 95 degree centigrade for 10 minutes in a PCR machine or by boiling to inactivate
Proteinase K completely.
5) Spin the tail lysate briefly before transferring to a PCR tube to exclude the tissue debris. Proceed
directly to PCR using the tail DNA lysate as a template at a volume rate of 1/10 as follows.
2. PCR reactions.
Contents of PCR mixture for wildtype/knockout allele screening:
5 ul tail DNA solution: spin briefly before transferring to a PCR tube to avoid contamination of debris.
1 ul 10 uM primers (each upper and lower primer)
5 ul 10x KOD dash DNA polymerase (from TOYOBO Co. LTD.,Japan)
5 ul 2.0 mM dNTPs
32 ul dd H2O
Total volume of 50 ul
We recently found that the final volume can be reduced to 25 ul without mineral oil application.
Sequences of PCR primers: should be designed according to your target gene.
Primers for detecting wild-type allele
Primers for detecting knock-out allele
Methods for Mouse Genotyping by PCR (protocol 2)
Transgenic Genotyping from Tail Biopsies
Harvard University--MCB Department / HSCI
Remove .5-1 cm of the tail and place in 1.5 ml Eppendorf tube. (Store at -20oC until ready to digest).
Digest in Lysis Buffer* + Proteinase K (to 200 ug/ml final conc.).
Incubate in 55oC water bath overnight. (Vortex 1x after 1-2 h).
Add .5 ml Phenol:Chloroform:Isoamyl alcohol (25:24:1) to each tube and vortex for 30 sec.
Spin at top speed in a microcentrifuge for 5 minutes.
Transfer upper (aqueous) phase to new tube; make sure no debris from the interface is transferred.
Add 1 ml of 100% EtOH.
Vortex briefly or shake. Stringy white precipitate (the genomic DNA) should now be visible.
Spin briefly (<1 min) just enough to get the DNA to cling to the plastic, and decant supernatant.
Wash with 1 ml of 70% EtOH.
Let air dry until the pellet becomes partially translucent, but do NOT over-dry, or the DNA will not go into solution any longer.
Redissolve the pellet in 100 ml TE, pH 8.0.
Check concentration, and calculate the total yield, which should be around 10 to 50 mg.
Use 100 ng for subsequent PCR analysis.
*Lysis Buffer:
10 mM Tris-HCl, pH 8.0
25 mM EDTA, pH 8.0
100 mM NaCl
0.5% SDS
DNA from Tail Biopsies
Genotyping Transgenic Rodents by PCR
Isolation of DNA from Mouse Tail Biopsies
Lac-Z Detection in Tail Biopsies
Preparation of Mouse Tail DNA for Dot Blots or PCR
Universal Mouse Genotyping Protocol Using PCR
beta globin Primers
lacZ Primers
neo Primers
PCR Primer Design Tools
PCR Troubleshooting
Ten Things That Can Kill Your PCR
Ten Things That Can Kill Your PCR. by Peter Frame. A blank PCR gel has got to be one of the most aggravating things about. molecular biology. ...www.mbi.ufl.edu/~rowland/protocols/pcr.htm
PCR trouble shooting, help, suggestions and advice
PCR trouble shooting, help, suggestions and advice. If your PCR amplification somehow performs unexpectedly, it is usually caused by one of the listed ...biologi.uio.no/bot/ascomycetes/PCR.troubleshooting.html
PCR Troubleshooting
Troubleshooting PCR. Polymerase Chain Reaction problems and solutions, PCR help.www.pcrstation.com/pcr-troubleshooting/
Troubleshooting Guide
MultiplexPCR Troubleshooting Guide. Poor amplification of some or all loci. Pipetting error /. reagents missing. Repeat experiment checking the ...
www.abgene.com/downloads/Guide_PCR-multiplex-v2-0208.pdf
PCR-Online.org - PCR Protocols, Troubleshooting and Information
Westernblotting.org: definitions, molecular biology links, protocols, troubleshooting and technical information for those interested in western blots and...
www.pcr-online.org/Troubleshooting.htm
PCR troubleshooting - MyBio
PCR troubleshooting - Web Resources. Optimizing DNA Amplification Protocols Optimizing DNA Amplification Protocols using the Eppendorf ?? Mastercycler ?? ...mybio.net/biowiki/PCR_troubleshooting
Troubleshooting PCR Why do I have non-specific bands when I run my ...
Appendix III:Troubleshooting. Successful PCR Guide. Takara Mirus Bio. 38. Causes.Trouble-shooting measures. Concentration of primers is too high ...www.takarabiousa.com/docs/PCR_TRBSHT.pdf -
Troubleshooting the PCR procedure Specific application of PCR ...
Troubleshooting the. PCR procedure. For a detailed discussion of the factors that. influence PCR and how to troubleshoot the ...www.roche-applied-science.com/PROD_INF/MANUALS/epitope/p18-19.pdf
Optimization and troubleshooting in PCR.
Optimization and troubleshooting in PCR. References. http://www.genome.org#References. This article cites 42 articles, 13 of which can be accessed free at: ...www.genome.org/cgi/reprint/4/5/S185.pdf -
EdgeBio ExcelaPure 96-Well UF PCR Purification Kit Troubleshooting ...
Troubleshooting Guide forExcelaPure 96-Well UF PCR Purification Kit>www.edgebio.com/tech/tsg/ExcelaPure96-wellUF_TSG.html
Ten Things That Can Kill Your PCR. by Peter Frame. A blank PCR gel has got to be one of the most aggravating things about. molecular biology. ...www.mbi.ufl.edu/~rowland/protocols/pcr.htm
PCR trouble shooting, help, suggestions and advice
PCR trouble shooting, help, suggestions and advice. If your PCR amplification somehow performs unexpectedly, it is usually caused by one of the listed ...biologi.uio.no/bot/ascomycetes/PCR.troubleshooting.html
PCR Troubleshooting
Troubleshooting PCR. Polymerase Chain Reaction problems and solutions, PCR help.www.pcrstation.com/pcr-troubleshooting/
Troubleshooting Guide
MultiplexPCR Troubleshooting Guide. Poor amplification of some or all loci. Pipetting error /. reagents missing. Repeat experiment checking the ...
www.abgene.com/downloads/Guide_PCR-multiplex-v2-0208.pdf
PCR-Online.org - PCR Protocols, Troubleshooting and Information
Westernblotting.org: definitions, molecular biology links, protocols, troubleshooting and technical information for those interested in western blots and...
www.pcr-online.org/Troubleshooting.htm
PCR troubleshooting - MyBio
PCR troubleshooting - Web Resources. Optimizing DNA Amplification Protocols Optimizing DNA Amplification Protocols using the Eppendorf ?? Mastercycler ?? ...mybio.net/biowiki/PCR_troubleshooting
Troubleshooting PCR Why do I have non-specific bands when I run my ...
Appendix III:Troubleshooting. Successful PCR Guide. Takara Mirus Bio. 38. Causes.Trouble-shooting measures. Concentration of primers is too high ...www.takarabiousa.com/docs/PCR_TRBSHT.pdf -
Troubleshooting the PCR procedure Specific application of PCR ...
Troubleshooting the. PCR procedure. For a detailed discussion of the factors that. influence PCR and how to troubleshoot the ...www.roche-applied-science.com/PROD_INF/MANUALS/epitope/p18-19.pdf
Optimization and troubleshooting in PCR.
Optimization and troubleshooting in PCR. References. http://www.genome.org#References. This article cites 42 articles, 13 of which can be accessed free at: ...www.genome.org/cgi/reprint/4/5/S185.pdf -
EdgeBio ExcelaPure 96-Well UF PCR Purification Kit Troubleshooting ...
Troubleshooting Guide forExcelaPure 96-Well UF PCR Purification Kit>www.edgebio.com/tech/tsg/ExcelaPure96-wellUF_TSG.html
Wednesday, September 10, 2008
About PCR
1. IntroductionIn 1983 Kary B. Mullis was driving through California on a moonlight night (Mullis, 1990). He was pondering how to use DNA polymerase with oligonucleotide primers in order to identify a given nucleotide at a given position in a complex DNA molecule, such as the human genome. During this drive he invented or discovered the elegant method of making unlimited DNA copies from a single copy of DNA, and called the method: "Polymerase Chain Reaction" (PCR). A couple of months later he conducted the first successful experiment. Ten years after his drive in California, he was awarded the Nobel Prize in Stockholm for his brilliant discovery (Carr, 1993).
PCR was first published in 1985 (Saiki et al., 1985) with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. (Erlich et al., 1991).
2. AnimationThe basic molecular events of PCR are illustrated in an animation of the liquid phase DNA amplification, which is a prerequisite of the solid phase DNA amplification. The whole animation can be seen in the DIAPOPS animation.
3. The basic reactionPCR is based on the recognition by a short piece of DNA (the primer) of a sequence on a larger, single stranded fragment of DNA (template strand). When the primer recognizes the template and binds (anneals) to the recognition sequence, the 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation). When the temperature is raised, the new DNA strand will melt away (denature) from the template, and the template is once again open for annealing of a new primer when the temperature is decreased. By adding a second primer which recognizes the template strand complementary to the first template, the elongation can proceed in the direction of the first primer. In the first round of elongation, this will ideally double the amount of template strands. In the second temperature cycling, half of the templates for the first primer will be new-synthesized fragments, all terminated where the second primer annealed. When these new fragments are recognized by the first primer, the elongation cannot proceed beyond the second primer, and the synthesized fragments will have a fixed length determined by the distance of the annealing sites of the two primers. New production of template strands take place in every temperature cycle. In this way the DNA sequence between the two primer sequences is amplified exponentially, yielding high concentrations of double-stranded DNA of the same length. The newly-formed double stranded DNA is denatured at 94-97ºC. Primers anneal at 35-72ºC (the exact temperature is primer- and assay dependent), and the new product is synthesized at 72ºC, which is the optimal temperature for the Taq-polymerase.
4. ConclusionPCR is capable of producing large amounts of DNA fragments from a single piece of template DNA as the amplification increases the amount of fragments produced exponentially. In theory, it is possible to detect a single copy of template DNA by PCR using simple methods. For this reason PCR is used to identify nucleic acid sequences that are only present in very small numbers in the sample to be analyzed.
Lecture of PCR-2
Introduction to PCR. Molecular biology relies on techniques that enable the detection or ... With the introduction of the Polymerase Chain Reaction (PCR), ...www.modares.ac.ir/elearning/mnaderi/Genetic%20Engineering%20course%20II/Pages/Lecture2.htm
PCR Technology
Introduction. Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a ...www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html
Introduction to PCR
Either way, the DNA is extracted from the source and is amplified via PCR (the Polymerase Chain Reaction). This allows very minute amounts of DNA to be ...nature.umesci.maine.edu/forensics/p_intro.htm
6.1 Polymerase Chain Reaction (PCR) Introduction6.1 Polymerase Chain Reaction (PCR). Introduction. T. he polymerase chain reaction technique employs oligonucleotide primers to amplify segments of ...www.fws.gov/policy/library/fh_handbook/Volume_1/Chapter_6.pdf
Real-Time PCR Introduction [M.Tevfik DORAK]
Overview by MT Dorak, University of Alabama at Birmingham, USA.dorakmt.tripod.com/genetics/realtime.html
YouTube - EDIROL PCR Introduction
This is a video introduction to our new PCR MIDI controllers.www.youtube.com/watch?v=vfiK7Fl75ZQ
PCR was first published in 1985 (Saiki et al., 1985) with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. (Erlich et al., 1991).
2. AnimationThe basic molecular events of PCR are illustrated in an animation of the liquid phase DNA amplification, which is a prerequisite of the solid phase DNA amplification. The whole animation can be seen in the DIAPOPS animation.
3. The basic reactionPCR is based on the recognition by a short piece of DNA (the primer) of a sequence on a larger, single stranded fragment of DNA (template strand). When the primer recognizes the template and binds (anneals) to the recognition sequence, the 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation). When the temperature is raised, the new DNA strand will melt away (denature) from the template, and the template is once again open for annealing of a new primer when the temperature is decreased. By adding a second primer which recognizes the template strand complementary to the first template, the elongation can proceed in the direction of the first primer. In the first round of elongation, this will ideally double the amount of template strands. In the second temperature cycling, half of the templates for the first primer will be new-synthesized fragments, all terminated where the second primer annealed. When these new fragments are recognized by the first primer, the elongation cannot proceed beyond the second primer, and the synthesized fragments will have a fixed length determined by the distance of the annealing sites of the two primers. New production of template strands take place in every temperature cycle. In this way the DNA sequence between the two primer sequences is amplified exponentially, yielding high concentrations of double-stranded DNA of the same length. The newly-formed double stranded DNA is denatured at 94-97ºC. Primers anneal at 35-72ºC (the exact temperature is primer- and assay dependent), and the new product is synthesized at 72ºC, which is the optimal temperature for the Taq-polymerase.
4. ConclusionPCR is capable of producing large amounts of DNA fragments from a single piece of template DNA as the amplification increases the amount of fragments produced exponentially. In theory, it is possible to detect a single copy of template DNA by PCR using simple methods. For this reason PCR is used to identify nucleic acid sequences that are only present in very small numbers in the sample to be analyzed.
Lecture of PCR-2
Introduction to PCR. Molecular biology relies on techniques that enable the detection or ... With the introduction of the Polymerase Chain Reaction (PCR), ...www.modares.ac.ir/elearning/mnaderi/Genetic%20Engineering%20course%20II/Pages/Lecture2.htm
PCR Technology
Introduction. Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a ...www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html
Introduction to PCR
Either way, the DNA is extracted from the source and is amplified via PCR (the Polymerase Chain Reaction). This allows very minute amounts of DNA to be ...nature.umesci.maine.edu/forensics/p_intro.htm
6.1 Polymerase Chain Reaction (PCR) Introduction6.1 Polymerase Chain Reaction (PCR). Introduction. T. he polymerase chain reaction technique employs oligonucleotide primers to amplify segments of ...www.fws.gov/policy/library/fh_handbook/Volume_1/Chapter_6.pdf
Real-Time PCR Introduction [M.Tevfik DORAK]
Overview by MT Dorak, University of Alabama at Birmingham, USA.dorakmt.tripod.com/genetics/realtime.html
YouTube - EDIROL PCR Introduction
This is a video introduction to our new PCR MIDI controllers.www.youtube.com/watch?v=vfiK7Fl75ZQ
Introduction to PCR
PCR—from (Dr. Chen, Dept of Biochem. & Mol. Biology, Univ. College London)
Polymerase Chain Reaction
1) Add the following to a microfuge tube:10 ul reaction buffer1 ul 15 uM forward primer1 ul 15 uM reverse primer1 ul template DNA5 ul 2 mM dNTP8 ul 25 mM MgCl2 or MgSO4 (volume variable)water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94C, 30-90 sec.b) annealing - 55C (or -5C Tm), 0.5-2 min. c) extension - 72C, 1 min. (time depends on length of PCR product and enzyme used)repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25C.
Note: Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work. d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP. e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots). f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.
BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.16.6 mM ammonium sulfate67.7 mM TRIS-HCl, pH 8.8910 mM beta-mercaptoethanol170 micrograms/ml BSA1.5-3 mM MgCl2
j) The PCR product may be purified using a number of commercially available products or by gel-purification if the template needed to be removed. It can also be sequenced.
k) Trouble shooting see Tavi's page, MycoSite, Alkami Biosystems, Promega and Sigma.
l) PCR methods
Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. A number of companies also produce hot start PCR products, See Alkami Biosystem.
"Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence.
Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.
Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the desired fragment is circularise by ligation, then PCR using primer complementary to the known sequence extending outwards.
AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).
RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. See also Quantiative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, Nested RT-PCR.
RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA. See also Gibco manual.
DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed.
Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens.
Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. Used to determint the amount of target template in the reaction.
Recusive PCR - Used to synthesise genes. Oligos used are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (~20 bases). Design of the oligo avoiding homologous sequence (>8) is crucial to the success of this method.
Asymmetric PCR
In Situ PCR
Mutagenesis by PCR
Far too many to list properly.
For more information, protocols and links, go to PCR jump station, Alkami Biosystem, Fermentas, Promega, and Sigma, See also PCR primer, PCR notes and PCR manual at Roche and Qiagen.
Other PCR links - PCR lectures, radio-labelled probes, Thermocycler suppliers
Polymerase Chain Reaction
1) Add the following to a microfuge tube:10 ul reaction buffer1 ul 15 uM forward primer1 ul 15 uM reverse primer1 ul template DNA5 ul 2 mM dNTP8 ul 25 mM MgCl2 or MgSO4 (volume variable)water (to make up to 100 ul)
2) Place tube in a thermocycler. Heat sample to 95C, then add 0.5 -1 ul of enzyme (Taq, Tli, Pfu etc.). Add a few drops of mineral oil.
3) Start the PCR cycles according the following schemes:
a) denaturation - 94C, 30-90 sec.b) annealing - 55C (or -5C Tm), 0.5-2 min. c) extension - 72C, 1 min. (time depends on length of PCR product and enzyme used)repeat cycles 29 times
4) Add a final extension step of 5 min. to fill in any uncompleted polymerisation. Then cooled down to 4- 25C.
Note: Most of the parameters can be varied to optimise the PCR (more at Tavi's PCR guide):a) Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.b) Template DNA concentration - PCR is very powerful tool for DNA amplification therefore very little DNA is needed. But to reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used, though too much template may increase the amount of contaminants and reduce efficiency.c) Enzymes used - Taq DNA polymerase has a higher error rate (no proof-reading 3' to 5' exonuclease activity) than Tli or Pfu. Use Tli, Pfu or other polymerases with good proof-reading capability if high fidelity is needed. Taq, however, is less fussy than other polymerases and less likely to fail. It can be used in combination with other enzymes to increase its fidelity. Taq also tends to add extra A's at the 3'end (extra A's are useful for TA cloning but needs to be removed if blunt end ligation is to be done). More enzymes can also be added to improve efficiency (since Taq may be damaged in repeated cycling) but may increase non-specific PCR products. Vent polymerase may degrade primer and therefore not ideal for mutagenesis-by-PCR work. d) dNTP - can use up to 1.5 mM dNTP. dNTP chelate Mg++, therefore amount of Mg++ used may need to be changed. However excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may therefore also reduce error rate. Larger size PCR fragment need more dNTP. e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation (note: store primers in small aliquots). f) Primer design - check primer sequences to avoid primer-dimer formation. Add a GC-clamp at the 5' end if a restriction site is introduced there. One or two G or C at the 3' end is fine but try to avoid having too many (it can result in non-specific PCR products). Perfect complementarity of 18 bases or more is ideal. See Guide.g) Thermal cycling - denaturation time can be increased if template GC content is high. Higher annealing temperature may be needed for primers with high GC content or longer primers (calculate Tm). Using a gradient (if your PCR machine permits it) is a useful way of determining the annealing temperature. Extension time should be extended for larger PCR products; but reduced it whenever possible to limit damage to enzyme. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min (also check suppliers' recommendations, actual rate is much higher). The number of cycle can be increased if the number of template DNA is very low, and decreased if high amount of template DNA is used (higher template DNA is preferable for PCR cloning - lower error rate in the PCR).
h) Additives -
Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol can protects Taq against heat damage, while formamide may lower enzyme resistence.
0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR / LA PCR). Perform a titration to determine to optimum concentration (1.3 M recommended). Reduce melting temperature (92 -93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5%DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits.
>50mM TMAC (tetramethylammonium chloride), TEAC (tetraethylammonium chloride), and TMANO (trimethlamine N-oxide) can also be used.
BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction.
See also Dan Cruickshank's PCR additives and Alkami Enhancers for more.
i) PCR buffer
Higher concentration of PCR buffer may be used to improve efficiency.
This buffer may work better than the buffer supplied from commercial sources.16.6 mM ammonium sulfate67.7 mM TRIS-HCl, pH 8.8910 mM beta-mercaptoethanol170 micrograms/ml BSA1.5-3 mM MgCl2
j) The PCR product may be purified using a number of commercially available products or by gel-purification if the template needed to be removed. It can also be sequenced.
k) Trouble shooting see Tavi's page, MycoSite, Alkami Biosystems, Promega and Sigma.
l) PCR methods
Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. A number of companies also produce hot start PCR products, See Alkami Biosystem.
"Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence.
Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.
Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the desired fragment is circularise by ligation, then PCR using primer complementary to the known sequence extending outwards.
AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).
RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. See also Quantiative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, Nested RT-PCR.
RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA. See also Gibco manual.
DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed.
Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens.
Q/C-PCR (Quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. Used to determint the amount of target template in the reaction.
Recusive PCR - Used to synthesise genes. Oligos used are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (~20 bases). Design of the oligo avoiding homologous sequence (>8) is crucial to the success of this method.
Asymmetric PCR
In Situ PCR
Mutagenesis by PCR
Far too many to list properly.
For more information, protocols and links, go to PCR jump station, Alkami Biosystem, Fermentas, Promega, and Sigma, See also PCR primer, PCR notes and PCR manual at Roche and Qiagen.
Other PCR links - PCR lectures, radio-labelled probes, Thermocycler suppliers
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