Notes on procedures and issues relating to restriction digests:
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.
iGEM - restriction digest protocol: http://partsregistry.org/Help:Protocols/Restriction_Digest
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
|Enzyme||Recognition site||Supplied buffer(s)||Time saver?||Heat sensitivity||Known working padding|
|BsrGI||T^GTACvA||NEBuffer 2, BSA||Yes|
|SfiI||GGCCNNNNNGGCC||Incubate at 50 C.|
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.
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|
|XhoI + EcoRI||NEBuffer 2-4|
|XhoI + BamH||NEBuffer 3|
|XhoI + PciI||NEBuffer 3|
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.
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.
Sequence Manipulation Suite: Restriction Digest: http://www.bioinformatics.org/sms2/rest_digest.html