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
EUR 512

Taq DNA Polymerase

9001-500
EUR 191

Maximo Taq DNA Polymerase

S101 500 units
EUR 46

Maximo Taq DNA Polymerase

S102 5x500 units
EUR 107

Maximo Taq DNA Polymerase

S103 20x500 units
EUR 328

Taq DNA polymerase (+dNTPs)

02-001 200U
EUR 153
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 257
Description: The Taq DNA polymerase (+dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA polymerase (-dNTPs)

02-011 200U
EUR 140
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 218
Description: The Taq DNA polymerase (-dNTPs) is available in Europe and for worldwide shipping via Gentaur.

Taq DNA Polymerase (500U)

9K-001-0001 500U
EUR 174.41

Taq DNA Polymerase (3000U)

9K-001-0002 3000U
EUR 485

Taq DNA Polymerase (6000U)

9K-001-0033 6000U
EUR 1028.75

Taq DNA Polymerase (1000U)

9K-001-0035 1000U
EUR 348.85

Taq DNA Polymerase (Recombinant)

abx073521-10000U 10000 U
EUR 592

Taq DNA Polymerase (Recombinant)

abx073521-1000U 1000 U
EUR 230

Taq DNA Polymerase (Recombinant)

abx073521-3000U 3000 U
EUR 328

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, 5units/㎕

T2200-050 500units
EUR 131

Taq DNA Polymerase, 5units/㎕

T2200-100 2x500 units
EUR 192

Taq DNA Polymerase, 5units/㎕

T2200-200 4x500 units
EUR 313

Taq DNA Polymerase (PCR)

G008 5000 U (1.0 ml)
EUR 205

Taq DNA Polymerase (PCR)

G009 1000 U (200 ul)
EUR 87

Taq DNA Polymerase (PCR)

G126 10000 U (2 x 1.0 ml)
EUR 309

Hotstart Taq DNA Polymerase

L7052001 1000 units
EUR 568
Description: Premade ready to use kits will always come in handy. Get your experiment done right form the first try by using a validated kit with perfectly balanced reagents proportions and compatibility and by following a clear protocol.

Hotstart Taq DNA Polymerase

L7052250 250 units
EUR 195
Description: Premade ready to use kits will always come in handy. Get your experiment done right form the first try by using a validated kit with perfectly balanced reagents proportions and compatibility and by following a clear protocol.

Champagne Taq DNA Polymerase

P122-d1 500 U (2.5 U/μl)
EUR 177

Champagne Taq DNA Polymerase

P122-d2 500 U (5 U/μl)
EUR 177

Champagne Taq DNA Polymerase

P122-d3 500 U (10 U/μl)
EUR 177

Taq Plus DNA Polymerase

P201-01 250 U
EUR 123

Taq Plus DNA Polymerase

P201-02 1000 U
EUR 178

Taq Plus DNA Polymerase

P201-03 3000 U
EUR 319

ACTaq? Taq DNA Polymerase, 1000U

E2100-1000U
EUR 217

ACTaq? Taq DNA Polymerase, 250U

E2100-250U
EUR 102

YEA taq DNA Polymerase wiodNTP

FYT011-500U 500U Ask for price

Fast-Taq DNA Polymerase (500U)

9K-001-0036 500U
EUR 350.54

Taq DNA Polymerase, 5u/ul

B0089(D0089) 5X200U
EUR 93.5

Taq Plus DNA Polymerase (Recombinant)

abx073522-2500U 2.500 U
EUR 592

Taq Plus DNA Polymerase (Recombinant)

abx073522-250U 250 U
EUR 230

Taq Plus DNA Polymerase (Recombinant)

abx073522-750U 750 U
EUR 328

Taq DNA Polymerase - 5,000 units

3245 1/EA
EUR 517

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

Anti-Taq DNA Polymerase Antibody

48369-100ul 100ul
EUR 333

Anti-Taq DNA Polymerase Antibody

48369-50ul 50ul
EUR 239

Recombinant (E.Coli) Taq DNA Polymerase

RP-394 1000 IU
EUR 164

Taq Plus DNA Polymerase (PCR)

G012 250 U (50 ul)
EUR 73

Taq Plus DNA Polymerase (PCR)

G040 1000 U (200 ul)
EUR 106

Genorise® Taq DNA Polymerase

GR108007 200 tests
EUR 230

Taq DNA Polymerase Polyclonal Antibody

7018-100
EUR 354

Taq DNA Polymerase Polyclonal Antibody

7018-30T
EUR 146

Taq DNA polymerase (TQ21) , DNA Aptamer, Biotinylated

AD-146-B Custom Ask for price

Taq DNA polymerase (TQ21) , DNA Aptamer, unlabeled

AD-146-U Custom Ask for price

Taq Plus DNA Polymerase, 5u/ul

D0090 200U
EUR 79

ACTaq? Taq Blue DNA Polymerase, 1000U

E2100B-1000U
EUR 227

ACTaq? Taq Blue DNA Polymerase, 250U

E2100B-250U
EUR 105

YEA taq DNA Polymerase wi dNTP

FYT001-500U 500U Ask for price

amfiSure Hot-Start Taq DNA Polymerase

P0322-010 100 units
EUR 147

amfiSure Hot-Start Taq DNA Polymerase

P0322-025 250 units
EUR 228

Cheetah Hot Start Taq DNA Polymerase

29050 500EU
EUR 296
Description: Minimum order quantity: 1 unit of 500EU

Taq DNA Polymerase, 5units/㎕ , Bulk Packing

T2200-B01 10,000 units
EUR 1157

Taq DNA Polymerase, 5units/㎕ , Bulk Packing

T2200-B05 5x10,000 units
EUR 5450

HiQTM Taq Red DNA Polymerase, 1unit/㎕

T2201-050 500units
EUR 131

HiQTM Taq Red DNA Polymerase, 1unit/㎕

T2201-100 2x500 units
EUR 192

HiQTM Taq Red DNA Polymerase, 1unit/㎕

T2201-200 4x500 units
EUR 313

Recombinant (E.Coli) Taq Plus DNA Polymerase

RP-395 250 U
EUR 164

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

Anti-Taq DNA Polymerase Conjugated Antibody

C48369 100ul
EUR 397

Taq DNA Polymerase (Mg2+ plus Buffer)

P101-01 1000 U
EUR 121

Taq DNA Polymerase (Mg2+ plus Buffer)

P101-02 5000 U
EUR 193

Taq DNA Polymerase (Mg2+ plus Buffer)

P101-03 10000 U
EUR 262

Taq DNA Polymerase (Mg2+ free Buffer)

P102-01 1000 U
EUR 121

Taq DNA Polymerase (Mg2+ free Buffer)

P102-02 5000 U
EUR 193

Taq DNA Polymerase (Mg2+ free Buffer)

P102-03 10000 U
EUR 262

Maximo DFS-Plus Taq DNA Polymerase DNA-free

N140 500 units
EUR 50

Maximo DFS-Plus Taq DNA Polymerase DNA-free

N142 5x500 units
EUR 120

Maximo DFS-Plus Taq DNA Polymerase DNA-free

N144 20x500 units
EUR 367

Taq DNA polymerase (TQ21) , DNA Aptamer, FITC labeled

AD-146-F Custom Ask for price

Taq DNA Polymerase (High Purity), 5u/ul

HTD0078 5X200U
EUR 108

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-010 100 units
EUR 150

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl3

P0330-020 2x100 units
EUR 239

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-050 5X100 units
EUR 388

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-250 25X100 units
EUR 1670

amfiXpand Taq DNA Polymerase,Including dNTP&MgCl2

P0330-500 50X100 units
EUR 2993

Taq DNA Polymerase with dNTP Mix (6000U)

9K-001-0018 6000U
EUR 1280.62

Taq DNA Polymerase with dNTP Mix (500U)

9K-001-0031 500U
EUR 292.73

Taq DNA Polymerase with dNTP Mix (3000U)

9K-001-0032 3000U
EUR 907.39

Taq DNA Polymerase with dNTP Mix (1000U)

9K-001-0034 1000U
EUR 432.37

Taq DNA Polymerase 2x Premix - 500 Reactions

3249 1/EA
EUR 190

Hot Start Taq DNA Polymerase - 1000 units

3293 1/EA
EUR 310

3G HotStart Taq DNA Polymerase (5u/ul)

3GHST81 5x500U
EUR 746.87

HotStart Taq DNA Polymerase (Mg2+ Plus Buffer)

RK20601 25 U Ask for price

Accuris Taq Polymerase

PR1000-1000 1 PC
EUR 172.66

Accuris Taq Polymerase

PR1000-500 1 PC
EUR 132.79

Accuris Taq Polymerase

PR1000-6000 1 PC
EUR 618.47

Accuris Taq Polymerase

PR1000-S 1 PC
EUR 69.79

Reversible 1-min Hot Start Taq DNA Polymerase

P950 - Ask for price

Reversible 1-min Hot Start Taq DNA Polymerase

P950L - Ask for price

Taq DNA Polymerase, Ultra Pure, 5 units/ul

T2016-001 1000 units
EUR 165

Taq DNA Polymerase, Ultra Pure, 5 units/ul

T2016-005 5000 units
EUR 300

Taq DNA Polymerase, Ultra Pure, 5 units/ul

T2016-010 10,000 units
EUR 583

Taq DNA Polymerase, Ultra Pure, 5 units/ul

T2016-100 100,000 units
EUR 2885

HiQTM Taq Red DNA Polymerase, 1unit/㎕ , Bulk Packing

T2201-B01 10,000 units
EUR 1157

HiQTM Taq Red DNA Polymerase, 1unit/㎕ , Bulk Packing

T2201-B05 5x10,000 units
EUR 5450

Genorise® CLT Taq DNA Polymerase (1000 U)

GR108003 1000 U
EUR 199

Taq DNA Polymerase (Mg2+ plus Buffer, with dNTP)

P101-d1 1000 U
EUR 136

Taq DNA Polymerase (Mg2+ plus Buffer, with dNTP)

P101-d2 5000 U
EUR 267

Taq DNA Polymerase (Mg2+ plus Buffer, with dNTP)

P101-d3 10000 U
EUR 409

Taq DNA Polymerase (Mg2+ free Buffer, with dNTP)

P102-d1 1000 U
EUR 136

Taq DNA Polymerase (Mg2+ free Buffer, with dNTP)

P102-d2 5000 U
EUR 267

Taq DNA Polymerase (Mg2+ free Buffer, with dNTP)

P102-d3 10000 U
EUR 409

DFS-"HOT" Taq DNA Polymerase (DNA-free sensitive, E-coli. DNA Free)

N150 500 units
EUR 102

DFS-"HOT" Taq DNA Polymerase (DNA-free sensitive, E-coli. DNA Free)

N152 5x500 units
EUR 394

DFS-"HOT" Taq DNA Polymerase (DNA-free sensitive, E-coli. DNA Free)

N154 20x500 units
EUR 1525

EZ Taq Polymerase (HotStart)

9K-005-0020 500U
EUR 204.86

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-025 250 unit
EUR 167

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-050 2X250 unit
EUR 250

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-100 4X250 Unit
EUR 421

amfiSure Taq DNA Polymerase, 1unit/ul, Including dNTP&MgCl2

P0310-250 10X250 Unit
EUR 739

Fast-Taq DNA Polymerase with 10mM dNTP Mix (500U)

9K-001-0003 500U
EUR 479.35

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.