Restriction Endonucleases From the Yeasen FuniCut™ Series, 5 minutes Digestion With Universal Buffer

Restriction Endonucleases From the Yeasen FuniCut™ Series, 5 minutes Digestion With Universal Buffer

Molecular cloning is used in nearly every laboratory. Restriction endonucleases are a crucial component in molecular cloning experiments, but people are also often bothered by various problems with restriction endonucleases experiments. Common problems are as follows:

  • There are so many types of restriction endonucleases, it is difficult to choose.
  • Miscleavage, random cleavage, or incomplete enzymatic cleavage.
  • The rate of digestion is sluggish; it may take one hour or even overnight.
  • Additionally, Multiple enzyme digestion experiments also need to choose a variety of enzyme digestion bTo

To prove with ide you a more thorough grasp of restriction endonucleases, the following will briefly explain what restriction endonucleases are and how various endonucleases are classified in answer to the first question. The Yeasen FuniCut™ series of Fast Restriction Endonucleases may readily resolve the second through fourth problems through strain modulation and an improved procedure!

 

1. What is Restriction Endonuclease?
2. Restrictions endonuclease nomenclature
3. Restrictions on endonuclease classification
4. Yeasen FuniCut™ Fast Restriction Endonucleases

 

1. What is Restriction Endonuclease?

Restriction endonucleases are a class of enzymes that can recognize specific nucleotide sequences in double-stranded DNA molecules and cut phosphodiester linkage in DNA chains at specific sites.

Different restriction endonucleases will recognize different DNA sequences, They can cut DNA inside the recognition sequence or at a place not far from the recognition sequence, resulting in various products, as illustrated in Figure 1. BamHI forms sticky ends, KpnI forms 3'ticky ends, and EcoRV forms blunt ends.

Figure 1. Schematic diagram of BamHI, KpnI, EcoRV digestion

 

2. Restrictions endonuclease nomenclature

The genus name, species name, bacterial strain or serotype, and the order of discovery are the primary factors used to name restriction endonucleases. The name guidelines are displayed in Table 1 below using BstEII as an example:

Table 1. Restrictions endonuclease nomenclature

Abbreviation

Full Name

Implication

B

Bacillus

First letter of genus name

st

stearothermophilus

The first two letters of the species name

E

ET

The first letter of the strain name

II

Second discovery

The order found in such bacteria

 

3. Restrictions on endonuclease classification

According to the complexity of the structure, the mode, of action and the difference between the cofactors, restriction enzymes can be divided into four categories. The following table 2 summarizes the characteristics and typical enzymes of each category.

Table 2. The variations among several restriction endonucleases

Endonuclease class

Features

Typical enzyme species

Type I

1. Recognition and modification cleavage.

2. It can recognize specific DNA sequences, and the digestion site is indefinitely away from the recognition site, up to thousands of bases.

3. Action necessitates ATP.

EcoB, EcoK, etc.

Type II

1. Only has the function of identifying cutting.

2. The recognition sequence is often a short Palindromic sequence (about 4–8 bp), and the specific digestion site of the restriction endonuclease is typically the recognized sequence.

3. Action necessitates Mg2+.

4. The type of restriction enzymes most frequently utilized in molecular cloning.

HindIII, NotI, etc.

Type III

1. Recognition and modification digestion.

2. Aboutseparatesp separate the recognition site from the digestion site.

3. Action necessitates ATP.

HinfIII et al.

Type IV

1. Cuts only methylated DNA sequences.

2. About 30 bp separate the recognition site from the digestion site.

McrA, McrBC, etc.

As indicated in Table 3 below, it may also be split into three groups based on the similarities and differences between the recognition and digestion sites.

Table 3. Comparative analysis of Isoschizomer, Neoschizomer, and Isocaudarner

 

Isoschizomer

Neoschizomer

Isocaudarner

recognition sequence

same

same

different

digestion sites

same

different

same

typical representative

AgeI and BshTI: both recognize and cleave 5′-A↓CCGGT-3′

SmaI (5′-CCC↓GGG-3′) and XmaI(5′-C↓CCGGG-3′)

BamHI (5'-G↓GATCC-3') and BglII (5'-A↓GATCT-3')

In view of the fact that some restriction endonucleases on the market still require overnight digestion, and the products are prone to incorrect digestion, Yeasen Fast Restriction Endoa nuclease, a universal buffer, complete accurate digestion in 5 minutes, allowing your digestion experiments no more troubles!

 

4. Yeasen FuniCut™ Fast Restriction Endonucleases

Yeasen FuniCut™ Fast Restriction Endonucleases

Figure 2. Yeasen FuniCut™ Fast Restriction Endonucleases

4.1 Features

  • Rapid digestion: the digestion can be finished in 5–15 minutes, which can be reduced by more than 1-2 hours compared with conventional digestion experiments.
  • Universal buffer: The multi-enzyme cleavage reaction is made simpler by the fact that any combination of endonucleases can use the same buffer.
  • Effect of enzyme digestion: comparable to N* and appropriate for its buffer.
  • Direct electrophoresis makes the process simpler by providing a red color buffer and allowing the digestion products to be delivered directly for electrophoresis.
  • Precise digestion: Even with overnight digestion, there is very little star activity.

4.2 Exhibition of performance

4.2.1 In FuniCut™ Buffer, single, double, and triple digestion takes place in 5 minutes.

Figure 3. Single, double, and triple digestion using the FuniCut Buffer can typically be finished in 5 minutes, for example, the triple digestion using EcoRI+KpnI+SmaI.

4.2.2 The impact of the enzyme digestion is comparable to that of N* and T*, and it works well with their buffers.

Figure 4. Compared to similar N*, T* goods, Yeasen FuniCut™ BamHI enzyme digesting effect is compatible with N*, T* brand buffers.

4.2.3 Overnight digestion with very low star activity

Figure 5. Utilize the rapid endonucleases HindIII, NheI, PstI, XbaI, and XhoI from the Yeasen, Tbydance with the experimental protocol advised by each brand. Perform an overnight (16 h) digestion and analyze the digested products using agarose gel electrophoresis.

4.2.4 Highly redundant and capable of handling some surplus substrate.

Figure 6. Using FuniCut™ Fast Restriction Endonucleases EcoR I, Eag I, XbaI, and Sac I, 1 μg and 1.5 μg of plasmid were used as substrates, respectively, and digested for 5 min, and the digested products were subjected to agarose gel electrophoresis.

Table 4. Products and Reaction Buffer Compatibility

Product name

Cat#

Recognition sequence

Temperature
(ºC)

Thermal deactivation temperature
(ºC)


FuniCut™ buffer

T* Buffer

N* Buffer

Ta* Buffer

Protected base (bp)

Responses

FuniCut™ AscI (inquire)

15001ES50

GG/CGCGCC

37

80

100

100

100

100

1

50T

FuniCut™ AvrII (inquire)

15002ES25

C/CTAGG

37

80

100

100

100

100

3

25T

FuniCut™ BamHI (inquire)

15003ES76

G/GATCC

37

80

100

100

100

100

3

500T

FuniCut™ BclI (inquire)

15004ES62

T/GATCA

37

80

100

100

100

100

3

125T

FuniCut™ BsaI (inquire)

15005ES50

GGTCTC(1/5)

37

80

100

100

100

100

Not
Determined

50T

FuniCut™ BstEII (inquire)

15006ES60

G/GTNACC

37

80

100

75

100

75

Not
Determined

100T

FuniCut™ ClaI (inquire)

15007ES50

AT/CGAT

37

80

100

100

100

100

4

50T

FuniCut™ DpnII (inquire)

15008ES50

/GATC

37

80

100

75

100

75

Not
Determined

50T

FuniCut™ EagI (inquire)

15009ES25

C/GGCCG

37

80

100

100

100

100

2

25T

FuniCut™ EcoRI (inquire)

15010ES78

G/AATTC

37

80

100

100

100

100

5

600T

FuniCut™ EcoRV (inquire)

15011ES70

GAT/ATC

37

80

100

100

100

100

5

200T

FuniCut™ HindIII (inquire)

15012ES76

A/AGCTT

37

80

100

75

100

100

3

500T

FuniCut™ HpaI (inquire)

15013ES50

GTT/AAC

37

80

100

100

100

50

1

50T

FuniCut™ KpnI (inquire)

15015ES70

GGTAC/C

37

80

100

100

100

100

2

200T

FuniCut™ MluI (inquire)

15016ES60

A/CGCGT

37

80

100

75

100

100

3

100T

FuniCut™ NcoI (inquire)

15018ES30

C/CATGG

37

80

100

100

100

100

3

30T

FuniCut™ NdeI (inquire)

15019ES70

CA/TATG

37

80

100

100

100

100

3

200T

FuniCut™ NheI (inquire)

15020ES30

G/CTAGC

37

80

100

100

100

100

3

30T

FuniCut™ NotI (inquire)

15021ES50

GC/GGCCGC

37

80

100

100

100

100

2

50T

FuniCut™ PstI (inquire)

15022ES76

CTGCA/G

37

No

100

100

100

100

2

500T

FuniCut™ SacI (inquire)

15023ES60

GAGCT/C

37

80

100

100

100

100

3

100T

FuniCut™ SalI (inquire)

15024ES70

G/TCGAC

37

80

100

100

100

100

3

200T

FuniCut™ SbfI (inquire)

15025ES25

CCTGCA/GG

37

80

100

100

100

100

2

25T

FuniCut™ SmaI (inquire)

15027ES60

CCC/GGG

37

80

100

100

100

100

1

100T

FuniCut™ Spel (inquire)

15028ES50

A/CTAGT

37

80

100

100

100

100

2

50T

FuniCut™ SphI (inquire)

15029ES50

GCATG/C

37

80

100

100

100

100

1

50T

FuniCut™ SspI (inquire)

15030ES56

AAT/ATT

37

80

100

50

100

100

2

60T

FuniCut™ StuI (inquire)

15031ES60

AGG/CCT

37

80

100

100

100

100

1

100T

FuniCut™ TaqI (inquire)

15032ES70

T/CGA

65

No

100

100

100

100

4

200T

FuniCut™ XbaI (inquire)

15033ES76

T/CTAGA

37

80

100

50

100

100

2

500T

FuniCut™ XhoI (inquire)

15034ES76

C/TCGAG

37

80

100

100

100

100

4

500T

FuniCut™ FspI (inquire)

15036ES50

TGC/GCA

37

80

100

100

100

100

3

50T

FuniCut™ HinfI (inquire)

15038ES76

G/ANTC

37

80

100

100

100

50

2

500T

FuniCut™ ApaLI (inquire)

15039ES70

G/TGCAC

37

80

100

75

100

100

4

200T

FuniCut™ NruI (inquire)

15040ES50

TCG/CGA

37

No

100

100

100

100

5

50T

FuniCut™ PacI (inquire)

15041ES25

TTAAT/TAA

37

80

100

100

100

100

2

25T

FuniCut™ PvuII (inquire)

15042ES70

CAG/CTG

37

No

100

100

100

100

4

200T

FuniCut™ SacII (inquire)

15043ES50

CCGC/GG

37

80

100

100

100

100

4

50T

FuniCut™ NsiI (inquire)

15044ES25

ATGCA/T

37

80

100

100

100

100

3

25T

FuniCut™ Esp3I (BsmBI) (inquire)

15048ES30

CGTCTC(1/5)

37

80

100

100

100

100

5

30T

FuniCut™ BglII (inquire)

15049ES60

A/GATCT

37

80

100

100

100

50

2

100T

FuniCut™ BstBI (inquire)

15050ES60

TT/CGAA

37

80

100

100

100

100

Not
Determined

100T

FuniCut™ MnlI (inquire)

15051ES50

CCTC(7/6)

37

80

100

100

100

100

2

50T

CpG: Affected by CpG methylation.

EK: Affected by EcoKI methylation.

EB: Affected by EcoBI methylation.

Dcm: Affected by Dcm methylation.

Dam: Affected by Dam methylation.