User:Jarle Pahr/Restriction: Difference between revisions
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{| class="wikitable" border="1" cellpadding="5" cellspacing="0" | {| class="wikitable" border="1" cellpadding="5" cellspacing="0" | ||
|- | |- | ||
! Enzyme!! Recognition site !! Supplied buffer(s) !! Time saver? !! Heat sensitivity | ! Enzyme!! Recognition site !! Supplied buffer(s) !! Time saver? !! Heat sensitivity !! Known working padding | ||
|- | |- | ||
| AgeI || ACCGGT || | | 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|| || || || | ||
|- | |- | ||
||BglI||GCCNNNNNGGC|| || || | |SfiI||GGCCNNNNNGGCC|| || ||Incubate at 50 C. || | ||
|- | |||
||BglI||GCCNNNNNGGC|| || || || | |||
|} | |} | ||
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| AgeI+PciI || NEBuffer 4||Have used NEBuffer 1. Seems to work reasonably well, but may lead to lower CIP activity if dephosphorylating. | | 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|| | |AgeI-HF + KpnI||NEBuffer 1|| | ||
|- | |- | ||
|BsrGI+BamHI-HF||NEBuffer 4|| | |BsrGI+BamHI-HF||NEBuffer 4|| |
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