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/

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

Enzyme	Recognition site	Supplied buffer(s)	Time saver?	Heat inactivation
AgeI	ACCGGT	Example
PciI	ACATGT	Example
EcoRI		Example
BsrGI	T^GTACvA	NEBuffer 2, BSA	Yes
SacII	CCGCGG	Nebuffer 4	No
NdeI	CATATG
BamHI	GGATCC

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

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