User:Jarle Pahr/Restriction

Notes on procedures and issues relating to restriction digests:

http://en.wikipedia.org/wiki/Restriction_digest

About star activity: https://www.neb.com/tools-and-resources/usage-guidelines/star-activity

Enzyme volume should not exceed 10 % of total reaction volume, due to glycerol content.

http://utminers.utep.edu/rwebb/html/restriction_digestion.html

http://www.molecularstation.com/dna/restriction-enzyme-digestion/

Protocols

iGEM - restriction digest protocol: http://partsregistry.org/Help:Protocols/Restriction_Digest

http://www.methods.info/Methods/RNA_DNA/restr_analysis.html

http://www.med.umn.edu/starrlab/prod/groups/med/@pub/@med/@starrlab/documents/content/med_content_370461.html

http://www.promega.com/resources/product-guides-and-selectors/restriction-enzyme-resource/restriction-enzyme-general-information/

Supercoiled plasmid DNA migrates faster than linear (ex cut plasmid) DNA: http://www.researchgate.net/post/Does_supercoiled_dna_migrate_faster_in_agarose_gel_electrophoresis_than_linear_form_of_dna

https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

Enzymes

Enzyme	Recognition site	Supplied buffer(s)	Time saver?	Heat sensitivity	Known working padding
AgeI	ACCGGT
KpnI	GGTACC
PciI	ACATGT		No
EcoRI	GAATTC
BsrGI	T^GTACvA	NEBuffer 2, BSA	Yes
SacII	CCGCGG	Nebuffer 4	No
NdeI	CATATG
BamHI	GGATCC
XhoI	CTCGAG		Yes
AflII	CTTAAG
SfiI	GGCCNNNNNGGCC			Incubate at 50 C.
BglI	GCCNNNNNGGC

Note that altough restriction enzyme recognition sites are often palindromic, the enzymes are generally NOT indifferent with respect to read direction (5'-3' vs 3'-5').

Example: GAATTC is an EcoRI recognition site. CTTAAG is NOT an EcoRI recognition site.

Double digests

NEB Double Digest finder: https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder

Double Digest tool: http://www.thermoscientificbio.com/webtools/doubledigest/

Enzyme combination	Buffer recommendation	Experience
AgeI+PciI	NEBuffer 4	Have used NEBuffer 1. Seems to work reasonably well, but may lead to lower CIP activity if dephosphorylating.
AgeI-HF + KpnI	NEBuffer 1
BsrGI+BamHI-HF	NEBuffer 4
XhoI + EcoRI	NEBuffer 2-4
XhoI + BamH	NEBuffer 3
XhoI + PciI	NEBuffer 3

Methylation

CpG: http://en.wikipedia.org/wiki/CpG_site

CpG methylation is catalyzed by CpG MTases found in higher eukaryotes. CpG methylation patterns are not retained if the DNA is cloned into a bacterial host.

See https://www.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation

According to the NEB site, most laboratory strains of E. coli contain the site-specific methylates Dam methylase, Dcm methylase and EcoKI methylase.

• Dam methylase: Methylates N6 of Adenine in the sequence GATC.
• Dcm methylase: Methylates C5 of cytosine in the sequences CCAGG and CCTGG.
• EcoKI methylase: Methylates adenine in the sequences AAC(N6)GTGC and GCAC(N6)GTT.

Dh5a is Dam+, meaning GATC sites will be methylated.

http://openwetware.org/wiki/E._coli_genotypes: "dam = adenine methylation at GATC sequences exist; high recombination efficiency; DNA repair turned on"

Exercise caution when planning cloning using enzymes sensitive to methylation: Performing cloning can cause a methylation site to overlap with the restriction site after the cloning.

Software

http://tools.neb.com/NEBcutter2/

http://rebase.neb.com/rebase/rebase.html

https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder

http://insilico.ehu.es/restriction/

Sequence Manipulation Suite: Restriction Digest: http://www.bioinformatics.org/sms2/rest_digest.html

http://molbiol-tools.ca/Restriction_endonuclease.htm