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  6. Invitrogen™ DNA Polymerase I, Large (Klenow) Fragment

Invitrogen™ DNA Polymerase I, Large (Klenow) Fragment

Catalog No. 18012039
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Description

Includes

DNA Polymerase I, Large (Klenow) Fragment is supplied with a vial of 10X REact™ 2 Buffer [500mM Tris-HCl (pH 8.0), 100mM MgCl2, 500mM NaCl], vial of dilution buffer.

DNA polymerase enzyme that lacks the 5' to 3' exonuclease activity of intact DNA Polymerase I, but does exhibit the 5' to 3' DNA polymerase and 3' to 5' exonuclease activities

Applications:
Fill-in of 5' overhangs . Synthesis of probes by random primers labeling method. Sequencing single- and double-stranded DNA. Site-directed mutagenesis.

Source:
Purified from E. coli expressing the Klenow fragment on a plasmid.

Performance and Quality Testing:
SDS-PAGE purity; single-stranded and double-stranded endodeoxyribonuclease and self-priming assays; performance evaluated in fill-in reaction.

Unit Definition:
One unit incorporates 10nMol of total deoxyribonucleotide into acid-precipitable material in 30 min. at 37°C using a template•primer.

Array CGH, Cloning, Gene Expression Analysis and Genotyping, Genotyping and Genomic Profiling, Restriction Enzyme Cloning

Order Info

Shipping Condition: Approved for shipment on Wet or Dry Ice

Specifications

Content And Storage DNA Polymerase I, Large (Klenow) Fragment is supplied with a vial of 10X REact™ 2 Buffer [500 mM Tris-HCl (pH 8.0), 100 mM
MgCl2 , 500 mM NaCl], vial of dilution buffer. Store at -20°C.
Description DNA Polymerase
Polymerase DNA Polymerase I
Exonuclease Activity 3' to 5'
Quantity 500 Units
Shipping Condition Approved for shipment on Wet or Dry Ice
Hot Start No

Frequently Asked Questions (FAQs)

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.
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.
Is there anything to prevent AmpliTaq Gold DNA polymerase from extending from the 3’ end of a TaqMan probe in a 5’ nuclease assay?

Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.

How does AmpliTaq Gold DNA Polymerase differ from AmpliTaq DNA Polymerase?

AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp 10X PCR Buffer I and/or GeneAmp 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

Does AmpliTaq Gold DNA Polymerase contain exonuclease (proofreading) activity?

No, AmpliTaq Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.
Does the fidelity of AmpliTaq DNA Polymerase change in the presence of base analogs?

The fidelity of this PCR enzyme is affected in two ways. First, AmpliTaq DNA Polymerase typically binds to and incorporates base analogs less efficiently than conventional dNTPs, which means that polymerase activity is lower in reactions that contain base analogs. Second, the analog may pair with more than one conventional complementary template base, so the analog may be incorporated at an increased level compared to conventional dNTPs. For the best fidelity, we recommend that base analogs are included at low concentrations in the reaction.
What is the expected half life of AmpliTaq DNA Polymerase at 95 degrees C?

The half-life of AmpliTaq DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.
What is "Hot-start" PCR?

Hot-start is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. The major obstacle to obtaining highly sensitive and specific amplifications appears to be competing side reactions such as the amplification of non-target sequences (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Mispriming can occur any time all components necessary for amplification are present at permissive temperatures (below optimal annealing temperature) such as during reaction set up. A hot start can be performed either manually or can be automated utilizing AmpliTaq Gold DNA Polymerase.

In the manual hot-start technique a key component necessary for amplification, such as the enzyme, is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. Once this temperature is reached, the missing component is added and the PCR amplification is allowed to proceed. Because a key component was withheld from the reaction at permissive temperatures, competing side reactions are minimized and specific amplification occurs.

AmpliTaq Gold DNA Polymerase facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase. Once activated, AmpliTaq Gold DNA Polymerase performs just as AmpliTaq DNA Polymerase does. Since it is provided in its inactive form, it can be added to a reaction without the fear of pre-PCR misprimed primers being extended. Once all of the components for amplification have been added to a tube, the reaction is heated to 95C for 5 - 10 minutes. This incubation activates the enzyme and allows the reaction to proceed normally.
How much MgCl2 should be added to the PCR amplification when using AmpliTaq DNA Polymerase or AmpliTaq Gold DNA Polymerase?

The standard starting point is a final concentration of 1.5 mM magnesium ion. Since each molecule of dNTP (total 0.8 mM per reaction at 200 µM each) binds a magnesium ion, 0.8 mM magnesium ions are unavailable for AmpliTaq DNA Polymerase to use; hence, 0.7 mM free magnesium ions will be available as a cofactor for Taq's polymerization activity. It is important to note that there are other substrates in PCR amplifications that can also bind free magnesium (such as primers and template) therefore, the magnesium ion concentration should be titrated in order to find the optimum concentration for each reaction.
How much AmpliTaq DNA Polymerase is used in a PCR amplification?

Most PCR amplifications use 2.5 units of AmpliTaq DNA polymerase per 100 µL reaction. A 25 µL reaction would use about 0.6 units. However, the optimal unit concentration per reaction should be empirically determined, and often the less is better rule applies. Using too much AmpliTaq may result in non-specific amplification.
When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?

The concentration of dNTPs in a standard PCR amplification is 200 µM each, for a total of 800 µM. This total dNTP amount corresponds to 39 µg of dNTPs. This is a huge excess and, when generating long PCR fragments, is not a limiting factor during the PCR amplification, as the amount of target DNA generated is generally no more than 1 µg. More importantly, the reaction condition variables need to be monitored more closely in order for successful long PCR amplification to occur.
What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?

Successful amplification of long PCR targets is dependent on variables such as sufficient extension time during the PCR amplification, cosolvent addition, pH of the reaction buffer, salt concentration, primer design, use of a hot start, DNA sample integrity, and the enzyme's proofreading and polymerase activities. A few examples of our long PCR enzymes include our Elonagase enzyme mix that can be used for amplicons up to 30kb (blend of Taq and proofreading enzyme) or our Phire Hot Start II enzyme mix that can be used for amplicons up to 20 kb (Taq polymerase). Read more here: https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/long-fragment-pcr.html

For Research Use Only. Not for use in diagnostic procedures.