User:Jarle Pahr/Restriction: Difference between revisions
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About star activity: https://www.neb.com/tools-and-resources/usage-guidelines/star-activity | 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. | Enzyme volume should not exceed 10 % of total reaction volume, due to glycerol content. | ||
http://utminers.utep.edu/rwebb/html/restriction_digestion.html | http://utminers.utep.edu/rwebb/html/restriction_digestion.html | ||
http://www.molecularstation.com/dna/restriction-enzyme-digestion/ | http://www.molecularstation.com/dna/restriction-enzyme-digestion/ | ||
=Protocols= | |||
iGEM - restriction digest protocol: http://partsregistry.org/Help:Protocols/Restriction_Digest | iGEM - restriction digest protocol: http://partsregistry.org/Help:Protocols/Restriction_Digest | ||
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http://www.methods.info/Methods/RNA_DNA/restr_analysis.html | 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= | |||
{| class="wikitable" border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! 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|| || || || | |||
|- | |||
|[http://rebase.neb.com/rebase/enz/BamHI.html 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/ | |||
{| class="wikitable" border="1" cellpadding="5" cellspacing="0" | {| class="wikitable" border="1" cellpadding="5" cellspacing="0" | ||
|- | |- | ||
! Enzyme!! | ! 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://tools.neb.com/NEBcutter2/ | ||
http://rebase.neb.com/rebase/rebase.html | 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 |
Latest revision as of 07:35, 15 May 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 | 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