RapidDigest AluI

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RapidDigest AluI
Cat No.    Digestion site    Quantity    Isoschizomer
RD1001    5’...A G ↓C T...3’
3’...T C ↑ G A...5’    60µl (1RDU/µl)    AluBI
Source:    Incubation      Inactive    Active site on λ DNA
Arthrobacter luteus     30 min at 37oC          20 min at 65oC    143

Supplied with:         300µl of 10X RapidDigest Universal Buffer,120 µl 10X RD Blue Buffer

Store at -20oC, avoid frequent thawing and freezing.
For in vitro use only
All RapidDigest Restriction Enzymes is completely active in Universal RD Buffer and digest DNA in 15-30 minutes or less.
RD restriction enzyme also eliminates need for sequential digestion during double digest methods.

Recommended assay
1-Add below materials to 0.5ml tube:

    Plasmid/ Lambda DNA    PCR product    Genomic DNA
Water DNase free    15ul    17ul         30ul
10X RapidDigest Buffer    2ul    2ul            5ul
DNA    2ul (up to 1ug)    10ul (˷0.2ug)    10ul (5ug)
RapidDigest Enzyme    1ul(1 RDU)    1ul(1 RDU)    5ul(5 RDU)
Total Volume    20ul    30ul    50ul

2- Mix gently and spin down.
3- Incubate at 37oC for 15-30 minutes1.
4- Inactive the enzyme by heating for 20 min at 65oC2.

1.    Time of incubation may need to optimization but it could be achieved between 5 to 30 minutes. For digestion of difficult to cleave-DNA, incubation time may extend to one hour.
2.    There are some alternative ways to stop the reaction:
-    Addition of EDTA pH 8.0 <0.5M> final 20mM.
-    Spin column DNA purification.
-    Agarose gel extraction.
-    Phenol- Chloroform extraction.
-    Ethanol precipitation.

Ligation and recutting:
After 5-fold over digestion with, >95% of the DNA fragments can be ligated and recut with this enzyme.

DNA Methylation:
No Inhibition: dam, CpG, dcm

Unit Definition:
One RapidDigest Unit (1 RDU) is the amount of enzyme required to completely digest 1µg of Lambda DNA in 15-30 minute in 1X RapidDigest buffer. 

Quality Control:
All preparations are assayed for contaminating endonuclease, 3'-exonuclease, 5'-exonuclease/

5'-phosphatase, as well as nonspecific single and double stranded DNase activities.

For double digestion simply add 1ul of each enzyme and scale up the reaction. For different temprature reaction start of the

enzymes that requires lower temprature.

λ DNA used as substrate for unit definition and quality control tests.