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Conventional Restriction Enzymes

SgeI

Product information

Reaction conditions

Recommended buffer for 100% activity	Optimal tempThermo Scientificure	Enzyme activity in Thermo Scieitnfic buffers, %						Tango™ buffer for double digestion
		B (blue) 1X	G (green) 1X	O (orange) 1X	R (red) 1X	Tango™ (yellow) 1X / 2X
SgeI	37°C	0-20	0-20	0-20	NR	NR	NR	NR

pBR322 DNA (dcm+)
1.4% agarose
6 cleavage sites

Conditions for 100% Activity

1X Buffer SgeI:
10 mM Tris-HCl (pH 8.0 at 37°C), 5 mM MgCl₂, 100 mM KCl, 0.02% Triton X-100 and 0.1 mg/ml BSA.
Incubate at 37°C.

Storage Buffer

SgeI is supplied in:
10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA and 50% (v/v) glycerol.

Ligation and Recleavage

After a 2-fold overdigestion with SgeI, more than 80% of the DNA fragments can be ligated and recut.

Digestion of Agarose-embedded DNA

Minimum 3 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded pBR322 DNA isolated from E.coli dcm+ strain DNA in 16 hours at 37°C.

Note

• DNA methylated by Dcm or CpG methyltransferases will be a substrate for SgeI.
• Greater than 3-fold overdigestion with SgeI may result in incomplete cleavage.
• At least two copies of SgeI recognition sequence are required for an efficient cleavage.
• Amount of the enzyme required for complete digestion of methylated DNA depends on the number of SgeI recognition sites. DNA cleavage products generated by target site cleavage facilitate the nonspecific cleavage by SgeI. Therefore, optimization of the enzyme amount is recommended for DNA cleavage.
• pBR322 DNA isolated from E.coli dcm+ strain (#SD0041) can be used as a DNA cleavage efficiency control. SgeI cleaves all six dcm methylated targets on pBR322 DNA.

Isoschizomers

Search for commercial isoschizomers using REsearch™.

Double Digestion

Perform double digestion using DoubleDigest™.

SgeI is not recommended for double digestion.

Methylation Effects

Dam: never overlaps – no effect.
Dcm: always cleaves DNA methylated by Dcm methyltransferase.
CpG: cleaves targets overlapping with CpG methylated sequences.
EcoKI: never overlaps – no effect.
EcoBI: never overlaps – no effect.

Methylation type	Sequence	Cleavage effect
Dcm (CCWGG)	Cm5CWGG	Cleaves only methylated DNA
CpG	5'...m5C GNG...3' 3'... Gm5CNC...5'	Cleaves only methylated DNA
CpG	5'...m5C Gm5C G...3' 3'... Gm5C Gm5C...5'	Cleaves only methylated DNA

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

DNA Digestion

We recommend digesting 0.2-1.5 µg DNA with a 2-fold to 10-fold excess of enzyme in a total volume of 20 µl. A typical restriction enzyme digestion protocol is below.

1. Add the following reaction components in the order indicated:

   Water, nuclease-free	16-16.5 µl
   10X recommended buffer for restriction enzyme	2 µl
   Substrate DNA	1 µl (~1 µg)
   Restriction enzyme	0.5-1 µl (5-10 u)
   Total volume	20 µl

2. Mix gently and spin down briefly.
3. Incubate at the optimal reaction temperature for 1-16 hours.

Note

• The digestion reaction may be scaled either up or down.
• Some enzymes require additional components to obtain the stated activity. In these cases, add the required additive and adjust the volume of water appropriately.

Digestion of PCR Products

The most convenient option for digestion of PCR-amplified DNA is the addition of a restriction enzyme directly to the reaction tube after completion of PCR. The majority of Fermentas restriction enzymes are active in Fermentas PCR buffers.
However, digestion of PCR products in the amplification mixture is often inefficient. Therefore, PCR reaction mixture should not make more than 1/3 volume of digestion reaction mixture to avoid inhibitory effects.

1. Add the following reaction components in the order indicated:

   PCR reaction mixture	10 µl (~0.1-0.5 µg of DNA)
   Water, nuclease-free	16-17 µl
   10X recommended buffer for restriction enzyme	2 µl*
   Restriction enzyme	1-2 µl (10-20 u)
   Total volume	30 µl

   * Only 2 µl of 10X reaction buffer is required for unpurified PCR product in a 30 µl reaction volume.
2. Mix gently and spin down briefly.
3. Incubate at the optimal reaction temperature for 1-16 hours.

Note

• For cloning applications, purification of PCR products prior to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the yield of ligation.
• If the restriction enzyme requires special additives (e.g., SAM), reduce the amount of water appropriately.
• If cleavage of the PCR product is inefficient purify the PCR products with the GeneJET™ PCR Purification Kit (#K0702) prior to digestion.
• After digestion, gel-purify the PCR product with the GeneJET™ Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513) to remove short DNA fragments which compete with the insert in a ligation reaction.

Double Digestion

DoubleDigest™ Engine
Our DoubleDigest™ engine is a great tool to find information on buffer and reaction conditions for your double digests. Simply select two restriction enzymes required for digestion, submit the query and follow the recommendations. The DoubleDigest™ engine is continuously updated with newly released Fermentas restriction enzymes.

Digestion of Agarose-embedded DNA

1. Embed the substrate DNA in 1% low melting temperature agarose, 1 µg / 30 µl.
2. Prepare ~30 µl agarose plugs with a GelSyringe® or similar agarose dispensing system.
3. Equilibrate the plug in 100 µl of the appropriate 1X restriction enzyme buffer for 15 min.
4. Place the plug in 100 µl of fresh 1X buffer containing the restriction enzyme (see Table "Digestion of Agarose-embedded DNA").
5. Incubate at 30-37°C for mesophilic enzymes or at 50-55°C for thermophilic enzymes for 4-16 hours.