User:Jarle Pahr/PCR: Difference between revisions

Revision as of 12:06, 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)

Polymerases

Polymerase	Speed (min/kb)	Fidelity	Supplier	Reference	5'-3' Exonuclease?	3'-5' exonuclease (proof reading)	Strand displacement?	Overhang?
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

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

http://mama.indstate.edu/pfaffle/ptaq/

http://www.ncbi.nlm.nih.gov/pubmed/23326812

Primers

Sequences for SLIC:

Name	Length (bp)	Sequence	Tm (C) [calculated]	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]	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	Tm (C) [calculated]	Tm (C) [Analytical]	GC (% / bp)	Comment
rrnB p1_74bp_FWD_R	caaccggtGTTGCGCGGTCAGAAAATTA
rrnB p1_74bp_REV_R	gtacatgtAGTGGTGGCGCATTATAGG
GreA_60bp_FWD_R	taaccggtGGCGCAACGCCCTATAAAGT
GreA_60bp_REV_R	gtacatgtATAGTCATTTTACCCTGAAGTTCCC
LacUV5_49bp_R_FWD	caaccggtGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCG
LacUV5_49bp_R_REV	gtacatgtTCCACACATTATACGAGCCGGAAGCATAAAGTGTA
ArgI_46bp_R_FWD	caaccggtGCTTTAGACTTGCAAATGAATAATCATCCATAT
ArgI_46bp_R_REV	gtacatgtTAAAATTCAATTTATATGGATGATTATTCATTT

Sequences for Ligation-independent cloning (LIC):

Name	Length (bp)	Sequence
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.