User:Jarle Pahr/Restriction

From OpenWetWare

Jump to: navigation, search

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/

Contents

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

Buffers

Enzymes

Enzyme Recognition site Supplied buffer(s) Time saver? Heat sensitivity Known working padding
AgeI ACCGGT
KpnIGGTACC
PciI ACATGT No
EcoRI GAATTC
BsrGIT^GTACvANEBuffer 2, BSA Yes
SacIICCGCGGNebuffer 4 No
NdeICATATG
BamHIGGATCC
XhoICTCGAG Yes
AflIICTTAAG
SfiIGGCCNNNNNGGCC Incubate at 50 C.
BglIGCCNNNNNGGC

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 4Have used NEBuffer 1. Seems to work reasonably well, but may lead to lower CIP activity if dephosphorylating.
AgeI-HF + KpnINEBuffer 1
BsrGI+BamHI-HFNEBuffer 4
XhoI + EcoRINEBuffer 2-4
XhoI + BamHNEBuffer 3
XhoI + PciINEBuffer 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

Personal tools