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
AgeI	ACCGGT	Example
PciI	ACATGT	Example
EcoRI	GAATTC	Example
BsrGI	T^GTACvA	NEBuffer 2, BSA	Yes
SacII	CCGCGG	Nebuffer 4	No
NdeI	CATATG
BamHI	GGATCC
XhoI	CTCGAG
AflII	CTTAAG
SfiI	GGCCNNNNNGGCC				Incubate at 50 C.

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.
BsrGI+BamHI-HF	NEBuffer 4
XhoI + EcoRI	NEBuffer 2-4
XhoI + BamH	NEBuffer 3
XhoI + PciI	NEBuffer 3

Methylation

CpG:

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"

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

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