Search

  • 联系我们
  • 货号直购

Exo-Klenow Fragment (cloned), DNA Polymerase I, 5 U/μL - FAQs

查看更多产品信息 Exo-Klenow Fragment (cloned), DNA Polymerase I, 5 U/μL - FAQs (AM2008)

5 个常见问题解答

我想将一个含5’或3’突出端的分子转变成平末端分子，应使用什么酶？这些酶有何区别？

T4 DNA聚合酶和大肠杆菌DNA聚合酶的Klenow片段，均可将突出端分子转变成平末端分子。但T4 DNA聚合酶的3’—5’核酸外切酶活性比Klenow更强。

What enzymes can I use to convert a molecule with a 5' or 3' overhang to a blunt molecule? What is the main difference between them?

T4 DNA polymerase and Klenow fragment of E.coli DNA polymerase can both convert overhangs to blunt molecules. The 3' - 5' exonuclease activity of the T4 DNA polymerase is much more efficient than Klenow.

Can regular Klenow be used for BioPrime Plus Array CGH labeling rather than exo-Klenow?

Regular Klenow may be used but is not recommended because yield, sensitivity, and reproducibility may be compromised compared to exo-Klenow. Exo-Klenow polymerase lacks both 5'-3#146; and 3#146;-5#146; exonuclease activity, producing higher yields of labeled sample than standard Klenow and thereby increasing sensitivity. Exo-Klenow polymerase also incorporates modified nucleotides more effectively than standard Klenow, enabling you to obtain stronger hybridization intensities and greater reproducibility of results. Another advantage with a simplified random priming protocol is that the protocol takes less than three hours.

Can exo-Klenow be purchased separately from your array labeling kits?

Yes, the catalog number for the exo-Klenow is AM2008. The enzyme concentration is 5 U/µL.

What conditions are optimal for fill-in reactions using Klenow (Large fragment of DNA Polymerase)?

Klenow can be used to "fill in" 5' overhangs of double-stranded DNA fragments using common restriction endonuclease buffers. We recommend REact 2 buffer (1X concentration is 50 mM Tris-HCl pH 8, 50 mM NaCl, 10 mM MgCl2) , but other buffers will also work.

Fill-in Reaction Conditions:

1. Dilute Large Fragment of DNA Polymerase I to 0.5 U/µL with Klenow Dilution Buffer.
2. To a 1.5-mL microcentrifuge tube on ice, add: 10X REact 2 Buffer - 3 µL, 0.5 mM dATP - 1 µL, 0.5 mM dCTP - 1 µL, 0.5 mM dGTP - 1 µL, 0.5 mM dTTP - 1 µL, DNA 0.5-1 µg, Large fragment of DNA Polymerase I - 1 µL, Autoclaved distilled water to 30 µL.
3. Mix gently and centrifuge briefly to bring the contents to the bottom of the tube.
4. Incubate at room temperature for 10-15 minutes or 20 minutes on ice.
5. Terminate fill-in reaction by phenol extraction.

To label the DNA fragment, use 1-2 µL of [alpha-32P]dNTP (400 Ci/mmol, 10 mCi/mL) (24-48 pmoles) instead of the corresponding cold dNTP.

Note: Thermo Fisher Scientific also offers an Exo minus Klenow, which is provided with its own buffers.