User:Jarle Pahr/PCR
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/
http://www.ncbi.nlm.nih.gov/pubmed/23326812
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 | 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 | 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 (C) [calculated] | Tm (C) [Analytical] | GC (% / bp) | Comment |
---|---|---|---|---|---|---|
rrnB p1_74bp_FWD_R | 28 | caaccggtGTTGCGCGGTCAGAAAATTA | ||||
rrnB p1_74bp_REV_R | 27 | gtacatgtAGTGGTGGCGCATTATAGG | ||||
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 |
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.
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://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?