User:Jarle Pahr/PCR

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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
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==Phusion polymerase==
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===Phusion polymerase===
Note on Phusion polymerase:
Note on Phusion polymerase:
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See https://www.neb.com/products/polymerases-and-amplification-technologies/polymerases-and-amplification-technologies/dna-polymerase-strand-displacement-activity
See https://www.neb.com/products/polymerases-and-amplification-technologies/polymerases-and-amplification-technologies/dna-polymerase-strand-displacement-activity
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===Home-grown polymerase===
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http://bitesizebio.com/articles/free-pcr-for-5-years-or-how-to-make-your-own-taq-and-pfu/
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http://www.openbiotech.com/product_p/popentaq.htm

Revision as of 14:09, 27 March 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:


https://eu.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/10/08/dna-oligonucleotide-resuspension-and-storage

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)


Contents

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 NoNo
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


http://bitesizebio.com/articles/mind-your-p%E2%80%99s-and-q%E2%80%99s-a-short-primer-on-proofreading-polymerases/


[Phusion polymerase]

Finnzyme Tm calculator for use with Phusion: http://www.thermoscientificbio.com/webtools/tmc/


Strand displacement:


See https://www.neb.com/products/polymerases-and-amplification-technologies/polymerases-and-amplification-technologies/dna-polymerase-strand-displacement-activity


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


Primers

Sequences for SLIC:

Name Length (bp) Sequence Tm (C) [calculated] Tm (C) [Analytical] GC (% / bp) Comment
rrnB p1_74bp_FWD40 agccgggcgatgccaaccggGTTGCGCGGTCAGAAAATTA For amplification of 74 bp promoter fragments plus SLIC linkers
rrnB p1_74bp_REV39 ctccattattattgtacatgAGTGGTGGCGCATTATAGG  ?
rrnB_p1_long_FWD20agccgggcgatgccaaccggGTATCCTACGCCCGTGGTTA? For amplification of 389 bp fragment plus SLIC linkers. Use together with rrnB p1_74bp_REV
GreA_60bp_FWD agccgggcgatgccaaccggGGCGCAACGCCCTATAAAGT  ?
GreA_long_FWD40agccgggcgatgccaaccggTCACCCTTAAGTACGCCGTT59 50.00For amplification of 399bp fragment plus SLIC linkers.
GreA_60bp_REV45 ctccattattattgtacatgATAGTCATTTTACCCTGAAGTTCCC?

Sequences for amplification from pSB-M1g:

Name Length (bp) Sequence Tm (C) [calculated] Tm (C) [Analytical] GC (% / bp) Comment
RBS-GFPstart20ATGGAGTCATGAACATATGG56 40For amplification from pSB-M1g, starting from RBS. Somewhat low GC content, and does not pass 5' end stability check in Clone Manager.
pSB-REV120TCAAGGATGTGGATCTGCTG57(2)/64.4(1)/57.3(3) 50For 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-REV220CCGGCTTTCTTAGACACTCT60.5(1) 50For 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-FWD20 CCAGATCACATGAAGCAGCA
GFP-END-REV TTTGTATAGTTCATCCATGCC
GFP-END-LVA-EcoRI-BamHI-REV72agaggatcccttaagttaagctactaaagcgtagttttcgtcgtttgctgcTTTGTATAGTTCATCCATGCC 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 Tm (C) [calculated] Tm (C) [Analytical] GC (% / bp) Comment
rrnB p1_74bp_FWD_RcaaccggtGTTGCGCGGTCAGAAAATTA
rrnB p1_74bp_REV_RgtacatgtAGTGGTGGCGCATTATAGG
GreA_60bp_FWD_RtaaccggtGGCGCAACGCCCTATAAAGT
GreA_60bp_REV_RgtacatgtATAGTCATTTTACCCTGAAGTTCCC
LacUV5_49bp_R_FWDcaaccggtGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG
LacUV5_49bp_R_REVgtacatgtTCCACACATTATACGAGCCGGAAGCATAAAGTGTA
ArgI_46bp_R_FWDcaaccggtGCTTTAGACTTGCAAATGAATAATCATCCATAT
ArgI_46bp_R_REVgtacatgtTAAAATTCAATTTATATGGATGATTATTCATTT


Sequences for Ligation-independent cloning (LIC):

Name Length (bp) Sequence Tm (C) [calculated] Tm (C) [Analytical] GC (% / bp) Comment
rrnB74bpLIC_FWD36gccgcgcggcagcctgGTTGCGCGGTCAGAAAATTA
rrnB75bpLIC_REV33caagaagaacccctAGTGGTGGCGCATTATAGG
GreA_60bpLIC_FWD36gccgcgcggcagcctgGGCGCAACGCCCTATAAAGT
GreA_60bpLIC_REV39caagaagaacccctATAGTCATTTTACCCTGAAGTTCCC
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.


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

Emulsion PCR

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024910

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

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://www.bio-rad.com/evportal/en/NO/evolutionPortal.portal?_nfpb=true&_pageLabel=SolutionsLandingPage&catID=LUSO3HC4S

http://originally4.blogspot.no/2011/08/pcr-for-species-with-extreme-gc-content.html

http://forums.biotechniques.com/viewtopic.php?f=2&t=14795

http://books.google.no/books?id=eY4VwJCJLmIC&pg=PA36&lpg=PA36&dq=PCR+pentamer+stability&source=bl&ots=7RpaJimjSl&sig=hwt2K1QO4JiOgiJzPZdtozVZaoE&hl=no&sa=X&ei=-mFGUdmDM4qHtQaX8oEg&ved=0CIwBEOgBMAk#v=onepage&q=PCR%20pentamer%20stability&f=false

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