Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.

A set of universal oligonucleotide primers for the retained regions of the eubactterial gene 16S arrnes has been designed for use with the 7700 Real-Time PCR Applied Biosystems (Taqman). During the development of this PCR, problems have been noted with the use of this gene as an amplification target. The contamination of reagents with bacterial DNA was a major problem exacerbated by the very sensitive nature of real-time PCR chemistry. This has been aggravated by the use of a small amplic of about 100 bases, as it is necessary with Taqman chemistry. In an attempt to overcome this problem, several methodologies have been applied. Some treatments were more efficient than others to eliminate contaminant DNA; However, to achieve this, there was a decrease in sensitivity.

With UV irradiation, a 4-log reduction of PCR sensitivity, with UV-facing 8-methoxypsorore activity, there was a 5 to 7 log discount, and with DNASE alone and in combination with a restriction digestion, There was a 1.66- reduction of the newspaper. Treatment of restriction endonuclease alone and together did not reduce the level of contaminant DNA. Without the development of Taq DNA polymerase ultrapide, ultrapular reagents and guaranteed plastic to be DNA free, the implementation of a PCR for the detection of the 16S Eubacterial 16S RNA with the Taqman system will continue to continue to be problematic. For three Taq polymerases from different commercial sources, the MICs were 0.08 to 0.64 micrograms of standard humic acids per ml and 0.24 to 0.48 micrograms of the moist acids coextraits per ml. The addition of the T4 Gene 32 protein increased the microphone for a 5.12 micrograms / ml taq.

Humic acids have decreased non-translative detection in DNA-DNA shoulder hybridizations at amounts of 0.1 micrograms and inhibited transformation of Escherichia coli coli HB101 with a plasmid with wide range of hosts, PUN1, at concentrations of 100 micrograms / ml. The purification of the gross DNA with ion exchange chromatography resulted in a 97% removal of the initially coextraits humic acids.

Quantity of triphosphates of cellular deoxynucleoside.

The eukaryotic cells contain a delicate scale of minus quantities of the four triphosphates of deoxyribonucleoside (DNTP), sufficient only for a few minutes of DNA replication. A deficiency and a single DNTP surplus can increase mutation rates, defective DNA repair or a depletion of mitochondrial DNA. DNTPs are generally quantified by an enzymatic test in which the incorporation of radioactive DATP (or radioactive DTTP into the DATP test) in synthetic oligonucleotides specific by a polymerase DNA is proportional to the concentration of the unknown DNTP. We find that the Klenow DNA polymerase commonly used can substitute the corresponding ribonucleotide for the unknown DNTP leading in some cases to a large overestimate of DNTPs.

We now describe the dosing conditions for each DNTP that avoid the constitution of ribonucleotides. For DTTP and DATP analyzes, it is sufficient to minimize the concentrations of the Klenow enzyme and marked DATP (or DTTP); For DCTP and DGTP, we had to replace Klenow’s enzyme with the polymerase of Taq DNA or thermal sequenease. We suggest that in some previous reports, the constitution of ribonucleotide can have caused too high values ​​for DGTP and DCTP. A new direct detection method of PCR amplified DNA in a closed system is described.

The method is based on the incorporation of primers labeled by energy transfer into the amplification product. PCR primers contain hairpin structures on their 5 years with fragments of donors and acceptors near the hairdressing rod. The primers are designed so that a fluorescent signal is generated only when the primers are incorporated into an amplification product.

Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.
Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.

Effects of amplification facilitators on diagnostic PCR in the presence of blood, fecal materials and meat.

The full potential of diagnostic PCR is limited, in part, through the presence of inhibitors in complex biological samples that reduce the efficiency of amplification. Therefore, various pre-PCR treatments are used to reduce the effects of PCR inhibitors. The objective of this study was to study the effects of 16 amplification facilitators to improve the amplification of DNA in the presence of blood, fecal or meat. Different concentrations of amplifying adapters and inhibitory samples were added to PCR mixtures containing RTTH or TAQ DNA polymerase. The addition of bovine serum albumin of 0.6% (WT / vol) on reaction mixtures containing DNA Taq polymerase reduced the inhibitory effect of blood and authorized the amplification of the DNA in the presence 2% instead of 0.2% blood (flight / vol). In addition, the addition of bovine serum albumin (BSA) with reaction mixtures containing faecal materials or meat has improved the amplification capabilities of the two polymerases.

The Taq DNA The polymerase was able to amplify the DNA in the presence of 4% instead of 0.4% (theft / vol) of faeces and 4% instead of 0.2% (flight / vol) , and RTTH was able to amplify the DNA in the presence of 4% instead of 0.4% (theft / vol) of feces and 20% instead of 2% meat (flight / vol). The T4 Gene T4 (GP32) protein (GP32) of the DNA bonded binding has presented an effect of relief similar to that of the BSA, except when it has been added to PCR mixtures of the meat containing and from The TAQ DNA Polymerase containing faeces. The effects of beetaine relief and a cocktail of proteinase inhibitors were more specific samples.

Taq DNA Polymerase

9001-2500 each
EUR 614.4

Taq DNA Polymerase

9001-500 each
EUR 229.2

Taq DNA Polymerase

L7051001 1000 unit
EUR 167

Taq DNA Polymerase

L7051200 200 unit
EUR 43

Taq DNA Polymerase

MB101-0500 500 U/100 μl
EUR 10

Taq DNA Polymerase

abx299725-100g 100 µg Ask for price

Taq DNA Polymerase

abx299725-20g 20 µg
EUR 337.5

Taq DNA Polymerase

abx299725-50g 50 µg Ask for price

Taq DNA Polymerase

abx461029-1096tests 10 × 96 tests
EUR 1787.5

Taq DNA Polymerase

abx461029-596tests 5 × 96 tests
EUR 1062.5

Taq DNA Polymerase

abx461029-96tests 96 tests
EUR 362.5

Taq DNA Polymerase (3000U)

9K-001-0002 3000U
EUR 582

Taq DNA Polymerase (6000U)

9K-001-0033 6000U
EUR 1234.5

Taq DNA Polymerase (1000U)

9K-001-0035 1000U
EUR 418.62

Taq DNA Polymerase (500U)

9K-001-0001 500U
EUR 209.29

TM Taq DNA Polymerase

TBS4010 1000 U
EUR 60

Taq HS DNA Polymerase

P132-d1 500 U
EUR 47.85

Taq HS DNA Polymerase

P132-d2 1,000 U
EUR 89.18

Taq HS DNA Polymerase

P132-d3 5,000 U
EUR 406

Taq DNA Polymerase (PCR)

G008 5000 U (1.0 ml)
EUR 246

Taq DNA Polymerase (PCR)

G126 10000 U (2 x 1.0 ml)
EUR 370.8

Taq DNA Polymerase Mix

abx461043-1096tests 10 × 96 tests Ask for price

Taq DNA Polymerase Mix

abx461043-596tests 5 × 96 tests
EUR 1062.5

Taq DNA Polymerase Mix

abx461043-96tests 96 tests
EUR 337.5

Taq DNA polymerase (-dNTPs)

02-011 200U
EUR 168
Description: The Taq DNA polymerase (-dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA polymerase (-dNTPs)

02-011-5 5×200U
EUR 261.6
Description: The Taq DNA polymerase (-dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA polymerase (+dNTPs)

02-001 200U
EUR 183.6
Description: The Taq DNA polymerase (+dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA polymerase (+dNTPs)

02-001-5 5×200U
EUR 308.4
Description: The Taq DNA polymerase (+dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA Polymerase, 5units/㎕

T2200-050 500units
EUR 157.2

Taq DNA Polymerase, 5units/㎕

T2200-100 2x500 units
EUR 230.4

Taq DNA Polymerase, 5units/㎕

T2200-200 4x500 units
EUR 375.6

Maximo Taq DNA Polymerase

S101 500 units
EUR 55.2

Maximo Taq DNA Polymerase

S102 5x500 units
EUR 128.4

Maximo Taq DNA Polymerase

S103 20x500 units
EUR 393.6

Hotstart Taq DNA Polymerase

L7052001 1000 units
EUR 595

Hotstart Taq DNA Polymerase

L7052250 250 units
EUR 167

Taq DNA Polymerase -1000 units

3243 1000units
EUR 115
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer

Champagne Taq DNA Polymerase

P122-d1 500 U (2.5 U/μl)
EUR 212.4

Champagne Taq DNA Polymerase

P122-d2 500 U (5 U/μl)
EUR 212.4

Champagne Taq DNA Polymerase

P122-d3 500 U (10 U/μl)
EUR 212.4

ACTaq? Taq DNA Polymerase, 1000U

E2100-1000U each
EUR 260.4

Taq DNA Polymerase - 5,000 units

3245 1/EA
EUR 449
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer

Recombinant Taq Dna Polymerase

27079806 EACH
EUR 607.62

Taq DNA Polymerase (Recombinant)

22060707-1 1,000 Unit(s)
EUR 137.27

Taq DNA Polymerase (Recombinant)

22060707-2 1500 Unit(s)
EUR 182.1

Taq DNA Polymerase (Recombinant)

22060707-3 5000 Unit(s)
EUR 465.77

Taq DNA Polymerase (Recombinant)

22060707-4 10 KU
EUR 840.96

Recombinant Taq DNA Polymerase

7-03634 1,000U Ask for price

Recombinant Taq DNA Polymerase

7-03635 3,000U Ask for price

Recombinant Taq DNA Polymerase

7-03636 10,000U Ask for price

Taq DNA Polymerase (Recombinant)

abx073521-10000U 10000 U
EUR 710.4

Taq DNA Polymerase (Recombinant)

abx073521-1000U 1000 U
EUR 276

Taq DNA Polymerase (Recombinant)

abx073521-3000U 3000 U
EUR 393.6

Taq DNA Polymerase Recombinant

rAP-1062 Inquiry Ask for price

ACTaq? Taq DNA Polymerase, 250U

E2100-250U each
EUR 122.4

Taq Plus DNA Polymerase

P201-01 250 U
EUR 147.6

Taq Plus DNA Polymerase

P201-02 1000 U
EUR 213.6

Taq Plus DNA Polymerase

P201-03 3000 U
EUR 382.8

Taq DNA Polymerase (5U/uL)

A1003-500U 500U
EUR 26.4
Description: Biotechnology

Taq DNA Polymerase, 5u/ul

B0089(D0089) 5X200U
EUR 112.2

Taq Plus DNA Polymerase (Taq Plus DNA) Protein

abx073522-100tests 100 tests
EUR 225

Taq DNA Polymerase (5U/µl), 2000U

PL1204 each Ask for price

Hot start Taq DNA Polymerase

EQ006 100μl/500U
EUR 200

Hot Start Taq DNA Polymerase

abx460010-1096tests 10 × 96 tests Ask for price

Hot Start Taq DNA Polymerase

abx460010-596tests 5 × 96 tests Ask for price

Hot Start Taq DNA Polymerase

abx460010-96tests 96 tests
EUR 262.5

Taq DNA Polymerase (5U/µl), 500U

PL1202 each Ask for price

Genorise® Taq DNA Polymerase

GR108007 200 tests
EUR 230

YEA taq DNA Polymerase wiodNTP

FYT011-500U 500U Ask for price

Taq DNA Polymerase (TaqDNA) Protein

abx073521-100tests 100 tests
EUR 225

Apex Red Taq DNA Polymerase, 10000u

42-402R 20 x 500u/Unit
EUR 1229.49
Description: 10X NH4 Buffer, Mg Free

Apex Red Taq DNA Polymerase, 500u

42-409R 1 x 500u/Unit
EUR 62.1
Description: 10X NH4 Buffer, Mg Free

Taq HS Red DNA Polymerase

abx461041-1096tests 10 × 96 tests Ask for price

Taq HS Red DNA Polymerase

abx461041-596tests 5 × 96 tests
EUR 2187.5

Taq HS Red DNA Polymerase

abx461041-96tests 96 tests
EUR 1062.5

Recombinant Taq Dna Polymerase - EACH

27079804 EACH
EUR 132.3

Recombinant (E.Coli) Taq DNA Polymerase

RP-394 1000 IU
EUR 196.8

Taq Plus DNA Polymerase (PCR)

G012 250 U (50 ul)
EUR 87.6

Taq Plus DNA Polymerase (PCR)

G040 1000 U (200 ul)
EUR 127.2

Taq DNA Polymerase Polyclonal Antibody

7018-100 each
EUR 424.8

Taq DNA Polymerase Polyclonal Antibody

7018-30T each
EUR 175.2

YEA taq DNA Polymerase wi dNTP

FYT001-500U 500U Ask for price

Taq DNA Polymerase, 5 units/µl

ABP-PP-ERTQ001 1000U Ask for price

Taq DNA Polymerase, 5 units/µl

ABP-PP-ERTQ100 100U Ask for price

Taq DNA Polymerase, 5 units/µl

ABP-PP-ERTQ200 200U Ask for price

Taq DNA Polymerase, 5 units/µl

ABP-PP-ERTQ500 500U Ask for price

Taq DNA Polymerase with Red Dye

abx461047-1096tests 10 × 96 tests
EUR 1787.5

Taq DNA Polymerase with Red Dye

abx461047-596tests 5 × 96 tests
EUR 1062.5

Taq DNA Polymerase with Red Dye

abx461047-96tests 96 tests
EUR 362.5

HotStart Taq DNA Polymerase (5U/uL)

A4144-250U 250U
EUR 66
Description: Biotechnology

ACTaq? Taq Blue DNA Polymerase, 1000U

E2100B-1000U each
EUR 272.4

Taq DNA Polymerase (Mg2+ Plus Buffer)

RK20600 5000U
EUR 49

Taq DNA Polymerase (Mg2+ plus Buffer)

P101-01 1000 U
EUR 37.29

Taq DNA Polymerase (Mg2+ plus Buffer)

P101-02 5000 U
EUR 231.6

Taq DNA Polymerase (Mg2+ plus Buffer)

P101-03 10000 U
EUR 290

Taq DNA Polymerase (Mg2+ free Buffer)

P102-01 1000 U
EUR 145.2

Taq DNA Polymerase (Mg2+ free Buffer)

P102-02 5000 U
EUR 231.6

Taq DNA Polymerase (Mg2+ free Buffer)

P102-03 10000 U
EUR 314.4

AtMax Taq DNA Polymerase (2.5U/µl), 200U

PL4201 each Ask for price

AtMax Taq DNA Polymerase (2.5U/µl), 500U

PL4202 each Ask for price

Chromo Taq DNA Polymerase (1U/µl), 200U

PL1205 each Ask for price

Chromo Taq DNA Polymerase (1U/µl), 500U

PL1206 each Ask for price

Taq DNA polymerase (TQ21) , DNA Aptamer, unlabeled

AD-146-U Custom Ask for price

ACTaq? Taq Blue DNA Polymerase, 250U

E2100B-250U each
EUR 126

Taq HS DNA Polymerase (Glycerol-free)

QL101-01 1,000 U
EUR 89

Taq HS DNA Polymerase (Glycerol-free)

QL101-02 5,000 U
EUR 406

Taq HS DNA Polymerase (Glycerol-free)

QL101-03 50,000 U
EUR 3625

Recombinant Taq Plus DNA Polymerase

7-03631 250U Ask for price

Recombinant Taq Plus DNA Polymerase

7-03632 750U Ask for price

Recombinant Taq Plus DNA Polymerase

7-03633 2,500U Ask for price

Taq Plus DNA Polymerase (Recombinant)

abx073522-2500U 2.500 U
EUR 710.4

Taq Plus DNA Polymerase (Recombinant)

abx073522-250U 250 U
EUR 276

Taq Plus DNA Polymerase (Recombinant)

abx073522-750U 750 U
EUR 393.6

Taq Plus DNA Polymerase Recombinant

rAP-1063 Inquiry Ask for price

Taq Plus DNA Polymerase, 5u/ul

D0090 200U
EUR 94.8

Taq DNA Polymerase, 5units/㎕ , Bulk Packing

T2200-B01 10,000 units
EUR 1388.4

Taq DNA Polymerase, 5units/㎕ , Bulk Packing

T2200-B05 5x10,000 units
EUR 6540

Taq DNA polymerase (TQ21) , DNA Aptamer, Biotinylated

AD-146-B Custom Ask for price

Taq DNA Polymerase M101 (5,000 U/mL)

RK26000 250U
EUR 60

amfiSure Hot-Start Taq DNA Polymerase

P0322-010 100 units
EUR 176.4

amfiSure Hot-Start Taq DNA Polymerase

P0322-025 250 units
EUR 273.6

DNA Amplification Kit with Taq DNA Polymerase, 100app

PL1202-K each Ask for price

Taq DNA Polymerase with Red Dye Mix

abx461048-1096tests 10 × 96 tests Ask for price

Taq DNA Polymerase with Red Dye Mix

abx461048-596tests 5 × 96 tests
EUR 1062.5

Taq DNA Polymerase with Red Dye Mix

abx461048-96tests 96 tests
EUR 325

The addition of 11.7% (WT / Vol) betaine has allowed the Taq DNA polymerase to amplify the DNA in the presence of 2% blood (theft / vol), while the addition of inhibitors Proteinase has led to an amplification of the DNA by the two polymerases in the presence of 4% (theft / vol) features. When different combinations of betaine, BSA, GP32 and proteinase inhibitors have been tested, no synergistic or additive effects have been observed. The effects of the facilitators on the synthesis of DNA in real time instead of conventional PCR have also been studied.