User:Jarle Pahr/Restriction: Difference between revisions
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CpG: | CpG: http://en.wikipedia.org/wiki/CpG_site | ||
Revision as of 10:13, 10 April 2013
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
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 |
---|---|---|---|---|
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. | ||
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. | |
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
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