User:Jarle Pahr/PCR: Difference between revisions
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^^ Tips on getting one band, by using one band of a first PCR as template for a second PCR: | ^^ Tips on getting one band, by using one band of a first PCR as template for a second PCR: | ||
PCR patent: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.htm&r=1&p=1&f=G&l=50&d=PTXT&S1=4,683,202.PN.&OS=PN/4,683,202&RS=PN/4,683,202 | |||
https://eu.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/10/08/dna-oligonucleotide-resuspension-and-storage | https://eu.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/10/08/dna-oligonucleotide-resuspension-and-storage | ||
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For a comparison of various polymerases, see http://barricklab.org/twiki/bin/view/Lab/ProtocolsTaq | For a comparison of various polymerases, see http://barricklab.org/twiki/bin/view/Lab/ProtocolsTaq | ||
http://www.biocompare.com/Life-Science-Videos/140397-How-to-Choose-the-Right-DNA-Polymerase-for-PCR/?catid=6539 | |||
===Phusion polymerase=== | ===Phusion polymerase=== | ||
Line 75: | Line 78: | ||
http://mama.indstate.edu/pfaffle/ptaq/ | http://mama.indstate.edu/pfaffle/ptaq/ | ||
http://www.ncbi.nlm.nih.gov/pubmed/23326812 | Optimization of Taq DNA polymerase enzyme expression in Escherichia coli.: http://www.ncbi.nlm.nih.gov/pubmed/23326812 | ||
Purification and characterization of Taq polymerase: A 9-week biochemistry laboratory project for undergraduate students.: http://www.ncbi.nlm.nih.gov/pubmed/21567784 | |||
==PCR programs== | ==PCR programs== | ||
Line 102: | Line 107: | ||
|rrnB_p1_long_FWD||20||agccgggcgatgccaaccggGTATCCTACGCCCGTGGTTA||?|| || ||For amplification of 389 bp fragment plus SLIC linkers. Use together with rrnB p1_74bp_REV | |rrnB_p1_long_FWD||20||agccgggcgatgccaaccggGTATCCTACGCCCGTGGTTA||?|| || ||For amplification of 389 bp fragment plus SLIC linkers. Use together with rrnB p1_74bp_REV | ||
|- | |- | ||
|GreA_60bp_FWD|| ||agccgggcgatgccaaccggGGCGCAACGCCCTATAAAGT || ?|| || || | |GreA_60bp_FWD||40 ||agccgggcgatgccaaccggGGCGCAACGCCCTATAAAGT || ?|| || || | ||
|- | |- | ||
|GreA_long_FWD||40||agccgggcgatgccaaccggTCACCCTTAAGTACGCCGTT||59|| ||50.00||For amplification of 399bp fragment plus SLIC linkers. | |GreA_long_FWD||40||agccgggcgatgccaaccggTCACCCTTAAGTACGCCGTT||59|| ||50.00||For amplification of 399bp fragment plus SLIC linkers. | ||
Line 125: | Line 130: | ||
|GFP-END-REV|| ||TTTGTATAGTTCATCCATGCC|| || || || | |GFP-END-REV|| ||TTTGTATAGTTCATCCATGCC|| || || || | ||
|- | |- | ||
|GFP-END-LVA-EcoRI-BamHI-REV||72||<sub>aga'''ggatcc'''''cttaag''ttaagctactaaagcgtagttttcgtcgtttgctgcTTTGTATAGTTCATCCATGCC</sub>|| || || ||Primer for amplification of end region of GFP from pSB-M1g with addition of LVA-tag plus EcoRI (italic) and BamHI (bold) sites. | |GFP-END-LVA-EcoRI-BamHI-REV||72||<sub>aga'''ggatcc'''''cttaag''ttaagctactaaagcgtagttttcgtcgtttgctgcTTTGTATAGTTCATCCATGCC</sub>||59.4(1) (target sequence) || || ||Primer for amplification of end region of GFP from pSB-M1g with addition of LVA-tag plus EcoRI (italic) and BamHI (bold) sites. | ||
|} | |} | ||
===Sequences for restriction-ligation cloning (RL cloning)=== | ===Sequences for restriction-ligation cloning (RL cloning)=== | ||
Line 135: | Line 137: | ||
{| class="wikitable" border="1" cellpadding="5" cellspacing="0" | {| class="wikitable" border="1" cellpadding="5" cellspacing="0" | ||
|- | |- | ||
! Name!! Length (bp) !! Sequence (PCR target sequence in bold)!! Tm (C) [calculated] | ! Name!! Length (bp) !! Sequence (PCR target sequence in bold)!!<sub>Tm, Target seq. (C) [calculated]</sub>!!GC (% / bp)!! Comment | ||
|- | |- | ||
|rrnB p1_74bp_FWD_R||28 ||caaccggt'''GTTGCGCGGTCAGAAAATTA''' | |rrnB p1_74bp_FWD_R||28 ||caaccggt'''GTTGCGCGGTCAGAAAATTA'''|| || || | ||
|- | |- | ||
|rrnB p1_74bp_REV_R||27||gtacatgtAGTGGTGGCGCATTATAGG|| || || | |rrnB p1_74bp_REV_R||27||gtacatgtAGTGGTGGCGCATTATAGG|| || || | ||
|- | |||
|rrnB P1_74bp_mutdis_REV|| ||gtacatgtAGTGGTGGTATATTATAGG|| || || | |||
|- | |- | ||
|GreA_60bp_FWD_R||28||taaccggt'''GGCGCAACGCCCTATAAAGT'''|| || || | |GreA_60bp_FWD_R||28||taaccggt'''GGCGCAACGCCCTATAAAGT'''|| || || | ||
|- | |- | ||
|GreA_60bp_REV_R||33||gtacatgtATAGTCATTTTACCCTGAAGTTCCC|| || || || | |GreA_60bp_REV_R||33||gtacatgtATAGTCATTTTACCCTGAAGTTCCC|| || || | ||
|- | |||
|LacUV5_49bp_R_FWD||43||caaccggtGCACCCCAGGCTT'''TACACTTTATGCTTCCGGCTCG'''||67.5(1)/60.7 (3) || || | |||
|- | |||
|LacUV5_49bp_R_REV||43||gtacatgtTCCACACATTATACGAGCCGGAAGCATAAAGTGTA|| || || | |||
|- | |||
|ArgI_46bp_R_FWD||41||caaccggtGCTTTAGACTTGC'''AAATGAATAATCATCCATAT'''||50.6(1)/44.7 (3) || || | |||
|- | |||
|ArgI_46bp_R_REV||41||gtacatgtTAAAATTCAATTTATATGGATGATTATTCATTT|| || || | |||
|- | |- | ||
| | |iraP_61bp_R_FWD||49||caaccggtGCTGGTAATCAAACAAAAAAT'''ATTTGCGCAAAGTATTTCC'''||59.7(1)/52.4(3)|| || | ||
|- | |- | ||
| | |iraP_61_bp_R_REV||48||gtacatgtAAGTATTATTTTTATGACAAAGGAAATACTTTGCGCAAAT|| || || | ||
|- | |- | ||
| | |LivJ_61_bp_R_FWD||48||caaccggtATTGTTAATAAACTGTCAAAA'''TAGCTATTCCAATATCATA'''||47.8(1)/44.2(3) || || | ||
|- | |- | ||
| | |LivJ_61_bp_R_REV||48||gtacatgtTGCTAAAACATACCCGATTTTTATGATATTGGAATAGCTA|| || || | ||
|- | |- | ||
| | |His_61bp_R_FWD||49||caaccggtGCCATAAAATATATAAAAAA'''GCCCTTGCTTTCTAACGTGAA'''||65.5(1)/59.1(3)|| || | ||
|- | |- | ||
| | |His_61bp_R_REV||49||gtacatgtGTCTTTTAACCTAAACCACTTTCACGTTAGAAAGCAAGGGC|| || || | ||
|- | |- | ||
| | |Thr_73bp_R_FWD||55||caaccggtAACTGGTTACCTGCCGTGAGTAAATT'''AAAATTTTATTGACTTAGGTC'''||51.7(1)/47.6(3) || || | ||
|- | |- | ||
| | |Thr_73bp_R_REV||55||gtacatgtTGCCTATATTGGTTAAAGTATTTAGTGACCTAAGTCAATAAAATTTT|| || || | ||
|- | |- | ||
| | |MazEF_60bp_R_FWD || ||caaccggtACGGGAGTTAGGCCGAAAT'''TTGCTCGTATCTACAATGTAG'''||55.2 (1) || || | ||
|- | |- | ||
| | |MazEF_60bp_R_REV|| ||gtacatgtTAGATACAGTATATATCAATCTACATTGTAGATACGAGCAA|| || || | ||
|- | |- | ||
| | |PcnB_50bp_R_FWD|| ||caaccggtTTGTAAATTCAACAT'''TCTCAAATGCGTCATGCTGA'''||66.1 (1) || || | ||
|- | |- | ||
| | |PcnB_50bp_R_REV|| ||gtacatgtGCGGCTAATCATAGCTCAGCATGACGCATTTGAGA|| || || | ||
|} | |} | ||
Line 223: | Line 235: | ||
[http://rnai.genmed.sinica.edu.tw/file/protocol/12_Sticky_end_PCRV2.pdf "sticky end PCR method"]: Can be used to generate PCR products with restriction site-compatible overhang. Can be used to clone fragments which contain the same restriction site(s) as the vector. | [http://rnai.genmed.sinica.edu.tw/file/protocol/12_Sticky_end_PCRV2.pdf "sticky end PCR method"]: Can be used to generate PCR products with restriction site-compatible overhang. Can be used to clone fragments which contain the same restriction site(s) as the vector. | ||
===LATE PCR=== | |||
http://www.smithsdetection.com/late-pcr.html | |||
http://www.pnas.org/content/101/7/1933.full.pdf | |||
===Colony PCR=== | ===Colony PCR=== | ||
Line 235: | Line 252: | ||
http://www.methodbook.net/pcr/pcrscreen.html | http://www.methodbook.net/pcr/pcrscreen.html | ||
Colony PCR can be performed with several amplification strategies: | |||
*Insert-specific primers only: For verifying presence of insert directly. Should only give amplicon if insert is present. However, if the insert was tranferred from one vector to another, colony PCR will give a positive result (amplicon) even if the vector is wrong | |||
*Backbone-specific primers: Altough the presence of the insert can't be confirmed directly in this way, it may still be useful to do colony PCR with primers specific to the regions in the vector flanking the insert, checking that the new vector gives the expected amplicon size. | |||
*Combination of insert- and backbone-specific primers: Useful in the case of short insert sequences. The presence of the insert can be verified directly while giving a longer, selectable amplicon size. Use the parent vector as a negative control template. | |||
===Emulsion PCR=== | ===Emulsion PCR=== | ||
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024910 | http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024910 | ||
===RT-PCR=== | |||
===qPCR=== | |||
===Digital PCR=== | |||
===Inverse PCR=== | |||
=PCR program design= | |||
Advantages of lower annealing temperature: | |||
*Possible higher yield | |||
*Shorter run time | |||
Disadvantages of lower annealing temperatures: | |||
*Higher danger of mis-priming and primer dimers | |||
*Longer run time. | |||
For short amplicons, a two-step cycling program may be tried. Given denaturation at 95 C and annealing at 50-55 C, the polymerase may have time to produce a product during the ramping phase from annealing to denaturation. | |||
==Touchdown PCR== | ==Touchdown PCR== | ||
Line 253: | Line 301: | ||
http://www.molecularinfo.com/MTM/E/E1/E1-3.html | http://www.molecularinfo.com/MTM/E/E1/E1-3.html | ||
http://peter.unmack.net/molecular/advice/primer.design.guide.html | |||
==Optimization and troubleshooting== | ==Optimization and troubleshooting== | ||
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Observations: For deliveries from Sigma Aldrich, Tm values from the Finnzymes Tm calculator and reported by Sigma are in good agreement. For deliveries from Macrogen, the reported Tm values systematically lower than the values calculated by Finnzymes Tm calculator - up to 6 degrees lower, and an average of 4,4 degrees lower. Does the synthesis process at Macrogen (impurities?) cause lower Tm values? | Observations: For deliveries from Sigma Aldrich, Tm values from the Finnzymes Tm calculator and reported by Sigma are in good agreement. For deliveries from Macrogen, the reported Tm values systematically lower than the values calculated by Finnzymes Tm calculator - up to 6 degrees lower, and an average of 4,4 degrees lower. Does the synthesis process at Macrogen (impurities?) cause lower Tm values? | ||
=Hardware= | |||
http://www.labome.com/method/PCR-Machines.html | |||
Comparison of PCR machines: | |||
EppenDorf MasterCycler | |||
OpenPCR | |||
PersonalPCR | |||
DIY designs: | |||
OpenPCR | |||
PersonalPCR | |||
http://www.scq.ubc.ca/MAKE/ | |||
ABI Prism 7700: http://www.ebay.com/itm/PERKIN-ELMER-ABI-PRISM-7700-SEQUENCE-DETECTOR-/300896880288?pt=LH_DefaultDomain_0&hash=item460eda06a0 | |||
See http://www.pcr-blog.com/files/tag-abi-prism-7700.html | |||
=Software= | |||
http://insilico.ehu.es/PCR/ | |||
http://genome.ucsc.edu/cgi-bin/hgPcr | |||
Primer3: http://sourceforge.net/projects/primer3/?source=directory | |||
PerlPrimer: | |||
=Links= | |||
http://www.pcrlinks.com/ |
Latest revision as of 13:33, 2 October 2013
http://nucleicacids.bitesizebio.com/articles/pcr-rescue/
^^ Tips on getting one band, by using one band of a first PCR as template for a second PCR:
http://www.spartanbio.com/wp-content/themes/spartan/assets/application_notes/17.pdf
For low-GC templates, some regions of the amplicon may have a melting temperature lower than the extension temperature, causing denaturation during the extension step and failure of the PCR reaction. (http://link.springer.com/article/10.1007%2Fs11274-010-0451-2?LI=true#page-1)
Polymerases
Polymerase | Speed (min/kb) | Fidelity | Supplier | Reference | 5'-3' Exonuclease? | 3'-5' exonuclease (proof reading) | Strand displacement? | Overhang? | Amplicon size |
---|---|---|---|---|---|---|---|---|---|
Taq | 1 | Example | ex | Yes | No | Yes | 3'-A | ||
Phusion | 0.25-0.50 | Example | NEB | https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530 | No | Yes | No | No | |
Pfu | 2 | Example | Example | No | Yes | No | |||
Q5 | ? | Example | NEB |
Polymerases: http://oregonstate.edu/instruction/bb492/lectures/DNAI.html
BiteSize Bio - The best polymerases of 2008: http://bitesizebio.com/articles/the-best-polymerases-of-2008/
For a comparison of various polymerases, see http://barricklab.org/twiki/bin/view/Lab/ProtocolsTaq
Phusion polymerase
Note on Phusion polymerase:
Quotes from the protocol:
The final concentration of each primer in a reaction using Phusion DNA Polymerase may be 0.2–1 μM, while 0.5 μM is recommended.
During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.
Annealing: Annealing temperatures required for use with Phusion tend to be higher than with other PCR polymerases. The NEB Tm calculator should be used to determine the annealing temperature when using Phusion. Typically, primers greater than 20 nucleotides in length anneal for 10–30 seconds at 3°C above the Tm of the lower Tm primer. If the primer length is less than 20 nucleotides, an annealing temperature equivalent to the Tm of the lower primer should be used. A temperature gradient can also be used to optimize
Finnzyme Tm calculator for use with Phusion: http://www.thermoscientificbio.com/webtools/tmc/
Strand displacement:
Home-grown polymerase
http://bitesizebio.com/articles/free-pcr-for-5-years-or-how-to-make-your-own-taq-and-pfu/
http://www.openbiotech.com/product_p/popentaq.htm
http://mama.indstate.edu/pfaffle/ptaq/
Optimization of Taq DNA polymerase enzyme expression in Escherichia coli.: http://www.ncbi.nlm.nih.gov/pubmed/23326812
Purification and characterization of Taq polymerase: A 9-week biochemistry laboratory project for undergraduate students.: http://www.ncbi.nlm.nih.gov/pubmed/21567784
PCR programs
JPAPHUS1:
SOE:
Primers
Sequences for SLIC
Name | Length (bp) | Sequence | Tm (C) [calculated] | Tm (C) [Analytical] | GC (% / bp) | Comment |
---|---|---|---|---|---|---|
rrnB p1_74bp_FWD | 40 | agccgggcgatgccaaccggGTTGCGCGGTCAGAAAATTA | For amplification of 74 bp promoter fragments plus SLIC linkers | |||
rrnB p1_74bp_REV | 39 | ctccattattattgtacatgAGTGGTGGCGCATTATAGG | ? | |||
rrnB_p1_long_FWD | 20 | agccgggcgatgccaaccggGTATCCTACGCCCGTGGTTA | ? | For amplification of 389 bp fragment plus SLIC linkers. Use together with rrnB p1_74bp_REV | ||
GreA_60bp_FWD | 40 | agccgggcgatgccaaccggGGCGCAACGCCCTATAAAGT | ? | |||
GreA_long_FWD | 40 | agccgggcgatgccaaccggTCACCCTTAAGTACGCCGTT | 59 | 50.00 | For amplification of 399bp fragment plus SLIC linkers. | |
GreA_60bp_REV | 45 | ctccattattattgtacatgATAGTCATTTTACCCTGAAGTTCCC | ? |
Sequences for amplification from pSB-M1g
Name | Length (bp) | Sequence | Tm (C) [calculated] | Tm (C) [Analytical] | GC (% / bp) | Comment |
---|---|---|---|---|---|---|
RBS-GFPstart | 20 | ATGGAGTCATGAACATATGG | 56 | 40 | For amplification from pSB-M1g, starting from RBS. Somewhat low GC content, and does not pass 5' end stability check in Clone Manager. | |
pSB-REV1 | 20 | TCAAGGATGTGGATCTGCTG | 57(2)/64.4(1)/57.3(3) | 50 | For amplification of vector backbone from pSB-M1g. Binds at same site as SeqMG1, between OriT and AgeI. Amplicon includes Colony PCR FWD2 site, but not the Seq5 site. | |
pSB-REV2 | 20 | CCGGCTTTCTTAGACACTCT | 60.5(1) | 50 | For amplification of vector backbone from pSB-M1g. Binds at beginning of SLIC linker B. Includes Colony PCR FWD2 and Seq5 sites, but not AgeI. Triggers primer dimer warning in Clone Manager. | |
GFP-END-FWD | 20 | CCAGATCACATGAAGCAGCA | ||||
GFP-END-REV | TTTGTATAGTTCATCCATGCC | |||||
GFP-END-LVA-EcoRI-BamHI-REV | 72 | agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgcTTTGTATAGTTCATCCATGCC | 59.4(1) (target sequence) | Primer for amplification of end region of GFP from pSB-M1g with addition of LVA-tag plus EcoRI (italic) and BamHI (bold) sites. |
Sequences for restriction-ligation cloning (RL cloning)
Name | Length (bp) | Sequence (PCR target sequence in bold) | Tm, Target seq. (C) [calculated] | GC (% / bp) | Comment |
---|---|---|---|---|---|
rrnB p1_74bp_FWD_R | 28 | caaccggtGTTGCGCGGTCAGAAAATTA | |||
rrnB p1_74bp_REV_R | 27 | gtacatgtAGTGGTGGCGCATTATAGG | |||
rrnB P1_74bp_mutdis_REV | gtacatgtAGTGGTGGTATATTATAGG | ||||
GreA_60bp_FWD_R | 28 | taaccggtGGCGCAACGCCCTATAAAGT | |||
GreA_60bp_REV_R | 33 | gtacatgtATAGTCATTTTACCCTGAAGTTCCC | |||
LacUV5_49bp_R_FWD | 43 | caaccggtGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG | 67.5(1)/60.7 (3) | ||
LacUV5_49bp_R_REV | 43 | gtacatgtTCCACACATTATACGAGCCGGAAGCATAAAGTGTA | |||
ArgI_46bp_R_FWD | 41 | caaccggtGCTTTAGACTTGCAAATGAATAATCATCCATAT | 50.6(1)/44.7 (3) | ||
ArgI_46bp_R_REV | 41 | gtacatgtTAAAATTCAATTTATATGGATGATTATTCATTT | |||
iraP_61bp_R_FWD | 49 | caaccggtGCTGGTAATCAAACAAAAAATATTTGCGCAAAGTATTTCC | 59.7(1)/52.4(3) | ||
iraP_61_bp_R_REV | 48 | gtacatgtAAGTATTATTTTTATGACAAAGGAAATACTTTGCGCAAAT | |||
LivJ_61_bp_R_FWD | 48 | caaccggtATTGTTAATAAACTGTCAAAATAGCTATTCCAATATCATA | 47.8(1)/44.2(3) | ||
LivJ_61_bp_R_REV | 48 | gtacatgtTGCTAAAACATACCCGATTTTTATGATATTGGAATAGCTA | |||
His_61bp_R_FWD | 49 | caaccggtGCCATAAAATATATAAAAAAGCCCTTGCTTTCTAACGTGAA | 65.5(1)/59.1(3) | ||
His_61bp_R_REV | 49 | gtacatgtGTCTTTTAACCTAAACCACTTTCACGTTAGAAAGCAAGGGC | |||
Thr_73bp_R_FWD | 55 | caaccggtAACTGGTTACCTGCCGTGAGTAAATTAAAATTTTATTGACTTAGGTC | 51.7(1)/47.6(3) | ||
Thr_73bp_R_REV | 55 | gtacatgtTGCCTATATTGGTTAAAGTATTTAGTGACCTAAGTCAATAAAATTTT | |||
MazEF_60bp_R_FWD | caaccggtACGGGAGTTAGGCCGAAATTTGCTCGTATCTACAATGTAG | 55.2 (1) | |||
MazEF_60bp_R_REV | gtacatgtTAGATACAGTATATATCAATCTACATTGTAGATACGAGCAA | ||||
PcnB_50bp_R_FWD | caaccggtTTGTAAATTCAACATTCTCAAATGCGTCATGCTGA | 66.1 (1) | |||
PcnB_50bp_R_REV | gtacatgtGCGGCTAATCATAGCTCAGCATGACGCATTTGAGA |
Sequences for Ligation-independent cloning (LIC):
Name | Length (bp) | Sequence | Tm (C) [calculated] | Tm (C) [Analytical] | GC (% / bp) | Comment |
---|---|---|---|---|---|---|
rrnB74bpLIC_FWD | 36 | gccgcgcggcagcctgGTTGCGCGGTCAGAAAATTA | ||||
rrnB75bpLIC_REV | 33 | caagaagaacccctAGTGGTGGCGCATTATAGG | ||||
GreA_60bpLIC_FWD | 36 | gccgcgcggcagcctgGGCGCAACGCCCTATAAAGT | ||||
GreA_60bpLIC_REV | 39 | caagaagaacccctATAGTCATTTTACCCTGAAGTTCCC | ||||
LacUV5_49bp_R_FWD | caaccggtGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG |
For SacII-based LIC sequence, see http://www.nmr.chem.uu.nl/users/rob/protocols/licdetailed.html
Tm notes:
- 1: Finnzyme/Thermo Scientific Tm Calculator
- 2: Clone Manager
- 3: Primer 3
BioBrick sequences:
dNTPs
Average molecular weight: 487 g/mol
Oligomer annealing
http://www.bio.net/bionet/mm/methods/1997-March/056072.html
http://www.protocol-online.org/biology-forums/posts/23721.html
http://www.oligos.com/annOligonucleotides.htm
http://www.addgene.org/plasmid_protocols/annealed_oligo_cloning/
PCR techniques
"sticky end PCR method": Can be used to generate PCR products with restriction site-compatible overhang. Can be used to clone fragments which contain the same restriction site(s) as the vector.
LATE PCR
http://www.smithsdetection.com/late-pcr.html
http://www.pnas.org/content/101/7/1933.full.pdf
Colony PCR
http://www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf
http://openwetware.org/wiki/Endy:Colony_PCR_protocol
https://www.researchgate.net/post/Protocol_for_colony_PCR
http://www.benchfly.com/video/57/how-to-perform-colony-pcr/
http://www.methodbook.net/pcr/pcrscreen.html
Colony PCR can be performed with several amplification strategies:
- Insert-specific primers only: For verifying presence of insert directly. Should only give amplicon if insert is present. However, if the insert was tranferred from one vector to another, colony PCR will give a positive result (amplicon) even if the vector is wrong
- Backbone-specific primers: Altough the presence of the insert can't be confirmed directly in this way, it may still be useful to do colony PCR with primers specific to the regions in the vector flanking the insert, checking that the new vector gives the expected amplicon size.
- Combination of insert- and backbone-specific primers: Useful in the case of short insert sequences. The presence of the insert can be verified directly while giving a longer, selectable amplicon size. Use the parent vector as a negative control template.
Emulsion PCR
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024910
RT-PCR
qPCR
Digital PCR
Inverse PCR
PCR program design
Advantages of lower annealing temperature:
- Possible higher yield
- Shorter run time
Disadvantages of lower annealing temperatures:
- Higher danger of mis-priming and primer dimers
- Longer run time.
For short amplicons, a two-step cycling program may be tried. Given denaturation at 95 C and annealing at 50-55 C, the polymerase may have time to produce a product during the ramping phase from annealing to denaturation.
Touchdown PCR
http://bitesizebio.com/articles/touchdown-pcr-a-primer-and-some-tips/
Hot-start
Primer Design
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
http://www.molecularinfo.com/MTM/E/E1/E1-3.html
http://peter.unmack.net/molecular/advice/primer.design.guide.html
Optimization and troubleshooting
http://cshprotocols.cshlp.org/content/2009/4/pdb.ip66.full
http://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/optimising_pcr/
http://www.protocol-online.org/biology-forums-2/posts/7644.html
http://www.protocol-online.org/biology-forums/posts/32105.html
http://europepmc.org/articles/PMC145803/pdf/241574.pdf
http://www.eppendorfna.com/int/index.php?l=131&action=products&contentid=109
http://originally4.blogspot.no/2011/08/pcr-for-species-with-extreme-gc-content.html
http://forums.biotechniques.com/viewtopic.php?f=2&t=14795
To do a quick, rough check on the thermocycling performance and corresponence between displayed and actual temperature, run a PCR program with a hold at 4 C at the end. Immediately after the display shows that 4C is reached and that the machine is holding the temperature, open the cover and feel the temperature of the heatblock. If it isn't cold, there is a significant time-lag/discorrespondence between the displayed and heatblock surface temperatures.
Purification of oligomers
Desalting: Standard purification procedure to remove by-products from synthesis.
Reverse-phase cartridge purification (Sigma:"RP1"). Separates truncated and full-length products on the basis of difference in hydrophobicity between full-length products with DMT protecting group present, and truncated sequences without DMT group. Unsuited for longer oligomers, as the proportion of DMT-containing truncated sequences increase with oligomer length. From Sigma website:
"As the oligo length increases, the proportion of uncapped products (truncated sequences bearing the DMT) tends to increase. These impurities will not be removed by RP1 and thus for longer oligos, HPLC or PAGE is recommended."
HPLC reverse-phase:
From Sigma website:
"The resolution based on lipophilicity will decrease with the length of the oligo. Therefore, RP-HPLC is usually not recommended for purifying products longer than 50 bases. Although longer oligos (up to 80 bases) can be purified using this method, the purity and yields may be adversely affected."
PAGE:
From Sigma website:
"This technique is recommended when a highly purified product is required. PAGE is the recommended purification for longer oligos (≥50 bases)."
Anion-Exchange HPLC:
From Sigma website:
"Anion- Exchange HPLC is limited by length (usually up to 40mers). The longer the oligonucleotide the lower the resolution on the Anion-Exchange HPLC column and thus the purity of the target oligo."
Comparison of calculated and reported Tm values
Oligomer | Sequence | Tm (C) [calculated] | Tm (C) [Analytical] | Supplier |
---|---|---|---|---|
GFP-END-FWD | 65.6(1) | 65.2 | Sigma | |
GFP-END-REV | 59.4(1) | 58.9 | Sigma | |
GFP-END-LVA-REV | 84.5(1) | 84.5 | Sigma | |
pSB-SeqA | TGCAAGAAGCGGATACAG | 60.7 | 60.2 | Sigma |
LacUV5_49bp_R_FWD | caaccggtGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG | 88.3(1) | 88.3 | Sigma |
LacUV5_49bp_R_REV | gtacatgtTCCACACATTATACGAGCCGGAAGCATAAAGTGTA | 78.2(1) | 78.1 | Sigma |
ArgI46bp_R_FWD | caaccggtGCTTTAGACTTGCAAATGAATAATCATCCATAT | 77.2(1) | 77.2 | Sigma |
ArgI46bp_R_REV | gtacatgtTAAAATTCAATTTATATGGATGATTATTCATTT | 67.8(1) | 67.6 | Sigma |
rrnB p1_74bp_FWD | agccgggcgatgccaaccggGTTGCGCGGTCAGAAAATTA | 91.2(1) | 91.3 | Sigma |
rrnB p1_74_bp_REV | ctccattattattgtacatgAGTGGTGGCGCATTATAGG | 75.7(1) | 75.6 | Sigma |
GreA_60bp_FWD | agccgggcgatgccaaccggGGCGCAACGCCCTATAAAGT | 91.5 | 91.6 | Sigma |
GreA_60bp_REV | ctccattattattgtacatgATAGTCATTTTACCCTGAAGTTCCC | 74.5 | 74.5 | Sigma |
LacUV5_49bp_FWD | AGCCGGGCGATGCCAACCGGgcaccccaggctttacactttatgcttccggctcg | 95.0(1) | 95.5 | Sigma |
LacUV5_49bp_REV | CTCCATTATTATTGTACATGtccacacaTTatacgagccggaagcataaagtgta | 80.3(1) | 80.3 | Sigma |
pJP-1 seq5 | CAGCGTGCGAGTGATTAT | 60.6(1) | 53.9 | Macrogen |
pJP-1 seq6 | AGACCACATGGTCCTTCT | 57.5(1) | 53.9 | Macrogen |
COPCR1FWD2 | TAATCGCCTTGCAGCACATC | 55.5(1) | 58.4 | Macrogen |
COPCR1REV | TTGCATCACCTTCACCCTCT | 65.1(1) | 58.4 | Macrogen |
SeqMG1 | AGCAGATCCACATCCTTGAA | 62.7(1) | 56.4 | Macrogen |
rrnB_p1_long_FWD | agccgggcgatgccaaccggGTATCCTACGCCCGTGGTTA | 90.6(1) | 85.1 | Macrogen |
GreA_long_FWD | agccgggcgatgccaaccggTCACCCTTAAGTACGCCGTT | 89.5(1) | 84.0 | Macrogen |
RF-LVA-EcoRI-FWD | GGGATTACACATGGCATGGATGAACTATACAAAGCAGCAAACGACGAAAACT | 84.0(1) | 80.5 | Macrogen |
Observations: For deliveries from Sigma Aldrich, Tm values from the Finnzymes Tm calculator and reported by Sigma are in good agreement. For deliveries from Macrogen, the reported Tm values systematically lower than the values calculated by Finnzymes Tm calculator - up to 6 degrees lower, and an average of 4,4 degrees lower. Does the synthesis process at Macrogen (impurities?) cause lower Tm values?
Hardware
http://www.labome.com/method/PCR-Machines.html
Comparison of PCR machines:
EppenDorf MasterCycler
OpenPCR
PersonalPCR
DIY designs:
OpenPCR
PersonalPCR
ABI Prism 7700: http://www.ebay.com/itm/PERKIN-ELMER-ABI-PRISM-7700-SEQUENCE-DETECTOR-/300896880288?pt=LH_DefaultDomain_0&hash=item460eda06a0
See http://www.pcr-blog.com/files/tag-abi-prism-7700.html
Software
http://genome.ucsc.edu/cgi-bin/hgPcr
Primer3: http://sourceforge.net/projects/primer3/?source=directory
PerlPrimer: