72kDa (Reducing)
10 mM Tris-HCl, 2 mM CaCl2 ,50% Glycerol，(pH 7.6, 25°C)
Store at -25 ~ -15℃
for 2 years
[1] Vanecko S, Laskowski M. Studies of the Specificity of Deoxyribonuclease I[J]. Journal of Biological Chemistry, 1961, 236(236):3312-6.
[2] Kienzle N, Young D, Zehntner S, et al. DNaseI treatment is a prerequisite for the amplification of cDNA from episomal-based genes[J]. Biotechniques, 1996, 20(4):612-6.
[3] Michael,R, Green, et
al. Human β-globin pre-mRNA synthesized in vitro is accurately spliced in
xenopus oocyte nuclei[J].Cell, 1983, 32(3):681-694.
DNase I
(Deoxyribonuclease I), can digest single or double-stranded DNA to produce mono
deoxynucleotides or single or double-stranded oligo deoxynucleotides, its
optimal working pH range is 7-8. DNase I activity is dependent on Ca2+
and can be activated by other bivalent metal ions such as Mg2+, Mn2+,
Zn2+, etc. In the presence of Mg2+, the enzyme can
randomly recognize and cut any site on any strand of DNA. In the presence of Mn2+,
two strands of DNA can be cut at the same site to form sticky ends with flat
ends or 1-2 nucleotides protruding.
Storage Solution: 2 U/ul DnaseⅠ、10mM Tris-Hcl、2mM CaCl2、50%Glycerol (pH7.6, 25℃)
10*Reaction
Buffer: 100mM Tris-Hcl、25mM MgCl2、5mM CaCl2 (pH7.6, 25℃)
This step is suitable for linearization of 1 μg DNA (≥100 nt) and can be scaled up according to experimental needs.
1）Add the following components in sequence
| Components |
Volume
|
Plasmid DNA
|
1μg DNA
|
10*Reaction Buffer
|
2μl
|
DnaseⅠ (2U/μl)
|
1μl
|
RNase-free ddH2O
| Up to 50μl |
2）Incubate at 37°C 1 h.
1. EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation 2. Please avoid repeated freeze-thaw cycles
The results of 1μg pBR322 plasmid digestion separated under different quantity of DnaseⅠ, The reaction was incubated for 10 minutes at 37°C, and 1% agarose gel was used for electrophoresis analysis after reaction.
M, marker;
Lane 1 1μg pBR322;
Lane 2 1μg pBR322 add 4U DNase I
Lane 3 1μg pBR322 add 2U DNase I
Lane 4 1μg pBR322 add 1U DNase I
Lane 5 1μg pBR322 add 0.5U DNase I
Lane 6 1μg pBR322 add 0.25U DNase I
Lane
7 1μg pBR322 add 0.125U DNase I
1μg (R: reducing condition, N: non-reducing condition).
您现在的位置：
