PCR - Miscellaneous - PCR - Miscellaneous

Uracil DNA Glycosylase (UDG), also called uracil N-glycosylase (UNG), catalyzes the release of uracil from uracil-containing DNA. The UDG/UNG from Genaxxon is a highly purified, recombinant enzyme isolated from E. coli with a mass of 26 kDa. UDG efficiently hydrolyzes single- and double-stranded DNA containing uracil, except for oligomers with 6 or fewer bases. Therefore, uracil-DNA glycosylase (UDG) is used wherever it is necessary to avoid the risk of DNA carryover (contamination) during PCR. In order to be able to hydrolyze DNA by UDG/UNG you do need dUTP > in your PCR reactions. Storage buffer: 20mM Tris HCl (pH 8.0) 50mM NaCl 0.1mM EDTA 50% Glycerin Reaction buffer (1X): 20 mM Tris HCl (pH 8.0) 1 mM EDTA

Pure, quality tested, nuclease-free water is suitable for use in all experiments that require nuclease-free water, including molecular biology applications. Nuclease-free water is processed, to yield DNase, RNase, and nuclease-free, deionized water. Contamination of water with nucleases may lead to inconsitencies or even complete failures of experiments. For this Genaxxon offers different grades of nuclease free water: sterile, Diethylpyrocarbonate (DEPC) treated water (M6082 >) and nuclease free water (not DEPC treated) (M6340 >). Our Nuclease-Free Water is provided in nuclease-free PE bottles or in glass bottles.

Sterile distilled water free of DNAses, Rnases and Proteinases. Water treated with DEPC to ensure absence of Rnase. For that reason this water is especially suited for RT-PCR or other molecular biology application where 100% absence of RNAse is necessary (resuspension of DNA, dilution of enzymes, etc.). Genaxxon offers different grades of nuclease free water: sterile, Diethylpyrocarbonate (DEPC) treated water (M6082 >) and nuclease free water (not DEPC treated) (M6340 >).

Oligo (dT)20 are single stranded oligodeoxyribonucleotides containing only deoxythymine (dT) to be able to prime with the poly(A) tail of mRNA molecules. Primers are quality controlled (MS), purified (reversed phase HPLC), aliquoted and lyophilized and came with a guaranteed amount of at least 5OD (about 27.2nmol (163.7µg)). Oligo (dT)20 Primer is designed to initiate the synthesis of cDNA from total RNA in a reverse transcription reaction, where Reverse Transcriptase, e.g. MMuLV (M3042) > is starting the reaction from the poly-A-end of mRNA. It can also be used for generation of labeled cDNA to screen microarrays. A mixture (1:1 ratio) of random hexamer primers (M3038 >) and Oligo(dT)20 primer may improve the sensitivity of cDNA synthesis as it will especially improve the efficiency of transcripts longer than 600 bp. Guaranteed delivery of at least 5OD (equals about 27.2nmol (163.7µg)). Primers are quality controlled (MS), purified (reversed phase HPLC), lyophilised and aliquoted. Dissolve content (5 OD) in 271.5μL of molecular biology grade water for an end concentration of 100 pmol/μL (100μM). 760μL (100μM) are sufficient for 760 reverse transcriptions of 20μL each. Oligo (dT)20 Primer is suitable for use as a primer for first strand cDNA synthesis with a reverse transcriptase, such as MMuLV or AMV. The primer hybridizes to the poly-adenylated tail found on the 3´ end of most eukaryotic mRNAs. Oligo (dT)20 ensures that the 3´ end of mRNAs are represented. Oligo(dT)20 Primer may be better suited for the new generation of Reverse Transcriptases like SuperScript™ that work at higher temperatures as the longer Oligo(dT)20 Primer enable annealing in reverse transcription reactions at higher temperatures. Applications• cDNA synthesis from total RNA in a reverse transcription reaction.• optimal choice for construction of cDNA libraries from eukaryotic mRNAs.• Full length cDNA cloning• 3’ rapid amplification of cDNA ends (3’ RACE)• Generation of labelled cDNA for screening of microarrays.Oligo (dT)20 Primer can not be used together with degraded RNA, prokaryotic RNA or miRNA (lack of poly(A) tail). Primer Sequence: 5´ – d (TTT TTT TTT TTT TTT TTT TT) –3´

Random Hexamers are short oligodeoxyribonucleotides of random sequence [d(N)6]. Primers are quality controlled (MS), purified (reversed phase HPLC), lyophilised and aliquoted and came with a guaranteed amount of at least 5OD (about 76.1nmol (136.4µg)). Hexanucleotide primers are a mixture of random 5'-hydroxyl hexanucleotides or hexamers, and can be used to quickly and efficiently prepare radioactive or non-radioactive probes using a DNA polymerase and a suitable DNA template or for cDNA synthesis from mRNA. The heterogeneous nature of the random primers ensures that all possible sequences will be represented in the probe mixture. As a result, these primers are well suited for generating as long as possible (complete) transcripts of the RNA, transcribing the 5 'ends, in particular for long RNA templates. As an alternative you can also use Oligo(dT)-Primer > for reverse transcription. Oligo(dT) primer bind to the polyA tail of RNA and therefore transcription will start always at the 3'-end of the RNA. The guaranteed delivery quantity of 5OD, corresponding to approx. 76.1nmol, or 136.4μg, can be dissolved in 760μL DEPC water to give a concentration of 100pmol/μL (100μM), or approx. 180μg/μL. If using between 50ng and 250ng of the hexamers per 20μL reverse transcription reaction, 760μL (100μM) will be sufficient for 460 to 2280 reverse transcriptase reactions.

SafeGel red stain is an ultra sensitive, extremely stable fluorescent dye designed to replace the toxic and possibly mutagenic ethidium bromide (EtBr) for staining dsDNA, ssDNA or RNA in agarose gels or polyacrylamide gels. SafeGel red stain is far more sensitive than EtBr without requiring a destaining step. SafeGel red stain is far less toxic and mutagenic compared to EtBr while both show virtually the same UV-spectra, so you can directly replace EtBr with SafeGel red stain without changing your existing imaging system. SafeGel can be used to stain dsDNA, ssDNA or RNA in agarose gel via either precast or post gel staining. SafeGel can also be used to stain dsDNA, ssDNA or RNA in polyacrylamide gel via post gel staining. SafeGel is also compatible with downstream DNA manipulations such as digestion with a restriction enzyme, Southern blotting techniques and clonings. A series of safety tests has confirmed that SafeGel is noncytotoxic, nonmutagenic and nonhazardous even at concentrations above the working concentrations used in gel staining. As a result, SafeGel can be safely disposed in regular trash, providing convenience and reducing cost in waste disposal. This fluorescent dye is supplied as a 10,000X solution in water. For customers who look for large pack size, we offer a cost-saving bulk pack size of 2mL, 5mL or 10mL (M3193.1010). Read in our blog why you should use Genaxxon's SafeGel now. For high-class agarose gels we offer the Genaxxon standard agarose LE > or our speciality agaroses for high resolution Tiny (M3046) > and Tiny HT (M3047) >.

Green DNA Dye is a sensitive fluorescent dye for detection of dsDNA in qPCR (real-time PCR) and other applications. Because Green DNA Dye has greater sensitivity for dsDNA, it is especially useful for assays where the presence of contaminating RNA or ssDNA might obscure results. With exceptionally low background fluorescence Green DNA Dye is ideal for use with laser scanners. In the detection of double-stranded DNA in native polyacrylamide gels it is also much more sensitive than silver staining. When Green DNA Dye is free in solution, it emits a very low fluorescent signal. As soon as the dye binds to the double-stranded DNA, the signal increases significantly (thousand fold), which makes the fluorescent signal of the dye directly proportional to the amount of amplified dsDNA. Green DNA Dye is delivered as 100-time solution in DMSO. The concentration of Green DNA Dye is traditionally given as a dilution factor where the 1X dilution is used for staining of DNA gels. For PCR, a 0.2X dilution is the starting dilution for optimization.  Our customers rely on Genaxxon's Green DNA Dye and use the dye for research into the regulation of tumor suppressor genes or vaccinations/disease patterns triggered by infections with enteroviruses, among other things. Preparation of a working solution for realtime PCR reactions:Green DNA Dye is suitable for real-time PCR with a suggested concentration for short targets of 0.2X (short targets: 50 – 400bp). Dilute delivered 100-time stock solution with nuclease-free PCR grade water by 1:10 (pre-dilution) and add 0.4µL of this solution to a 20µL final volume (PCR reaction). The 10-time working solution has to be kept at -20°C and has to be renewed every 2-3 weeks latest. Green DNA dye is a low toxic fluorescent nucleic acid dye as an alternative to ethidium bromide. Are you looking for more non-toxic dyes for your gel electrophoresis? Then try SafeGel red stain or SafeGel green stain - the cost-effective alternatives to EtBr. For qPCR we offer various qPCR Master Mixes > for your needs, with or without ROX, e.g., Green Master Mix High ROX >, Green Master Mix No ROX > or Probe Master Mix No ROX for qPCR >. Online form for your qPCR master mix test sample >

ROX is used as passive reference dye to compensate for non-PCR related well to well variations in the fluorescence. Variations in fluorescence can occur e.g. due to well to well variation in light intensity, which depends on the optical construction of the PCR machine or due to pipetting variations. The ROX fluorescence does not change during the course of the PCR reaction but provide a stable baseline to which samples are normalized. The excitation and emission of the reference dye are 584nm and 612nm, respectively.The ROX concentration to be used depends on the Real Time PCR instrument or, more precisely, on the filter available. On earlier Real Time PCR machines there was no filter matching precisely to the ROX fluorescence. Therefore, high ROX concentrations are needed with these machines. Some newer machines do not need any internal standard to correct for light intensity. Nevertheless, an internal standard might still be useful to correct for pipetting variations.This dye has been qualified for use on instruments like the ABI PRISM® 7700, One-Step System, RotorGene and many others.Please consult the user manual of the real time PCR instrument with regard to appropriate levels of ROX. Ready to use qPCR Master Mixes with High ROX (500nM) >, Low ROX (50nM) > or without ROX > are also available from Genaxxon.

The better performance of the Genaxxon bioscience 5X B-Enhancer Solution compared to standard enhancers such as form amide, DMSO, TMCA or BSA especially when used with GC rich regions or templates with a high degree of secondary structures depends on its ability to lower the DNA melting temperature and its enhancing effect on the DNA polymerase. In detail, Betaine binds preferentially to AT rich sequences in the major groove, thereby stabilizing AT rich regions of the DNA. Because AT forms 2 hydrogen bonds and GC forms 3, the bonding of AT is less stable than the one of GC. As a consequence of the stabilizing effect of Betaine on AT bonding, the stability of AT bonding and GC bonding is brought close to an equal level. At the same time, Betaine has a sequence independent destabilizing effect on all DNA. Summarized, the Tm of AT rich and GC rich sequences are equalized and the overall Tm is lowered. Furthermore, Betaine aids the processivity of thermostable polymerases and reduces "pauses" in polymerization caused by secondary structure that can induce the polymerase to disassociate from the DNA strand.As Betaine has a decreasing effect on the melting temperature of DNA and primers the denaturation temperatures as well as primer annealing temperatures should be reduced by 1 – 5°C compared to the annealing temperature already used. The optimal annealing temperature should be determined individually for each reaction.

Loading buffer I Fluoro is a fluorescent reagent that produces instant visualization of DNA bands upon blue light or UV illumination of agarose gels. Supplied in 6X DNA Loading Buffer, Loading buffer I Fluoro is used to prepare DNA markers or samples for loading on agarose or polyacrylamide gels.  Loading buffer I Fluoro is the sensitive staining reagent available for detecting the double-stranded DNA (dsDNA). It contains three tracking dyes (bromophenol blue, xylene cyanol FF, and orange G) for visually tracking the DNA migration during the electrophoresis plus an additional fluorescence dye for detection of DNA bands without an additional staining of the gel. It is an ideal alternative to Ethidium Bromide (EtBr). Approximate fluorescence excitation / emission: 300, 495 / 537 nm, bound to nucleic acid. For more information on our SimplyEnlight PCR products, read our blog now. Genaxxon's SimplyEnlight PCR - Unlock the Power of Your PCR! Picture 1: Comparison of UV- and blue light excitation             Picture 2: Effect of EtBr plus UV on cloning efficiency.

Gel Loading Dye I 6X is a loading buffer for DNA application on gels. Buffer contains Glycerol, EDTA, Bromophenol Blue and Xylene cyanol. Genaxxon 6X DNA Loading Dye is used to prepare DNA markers and samples for loading on agarose or polyacrylamide gels. It contains two different dyes (bromophenol blue and xylene cyanol FF) for visual tracking of DNA migration during electrophoresis.

DNA 6X Loading buffer is a special loading buffer for DNA application on gels that enables viusalization of DNA bands smaller than 100 bp without problems. Buffer contains Glycerol, EDTA, Orange G. The special dye gives the opportunity to detect even DNA bands running below 65 bp. Other loading dye: M3308 and PCR dye: M3309.

Coral Red Buffer is a 10-time dye solution as supplement for PCR buffers. This additive contains Glycerol, EDTA, a red and a yellow dye. 5µL of this solution are added to 45µL of the PCR assay before the PCR reaction to stain it red. An alternative to this product is the Genaxxon bioscience ready-to-use RedMastermix M3029. The RedMastermix contains all incredients necessary for PCR including a red dye that enables to control the different pipetting steps.

5X PCR Buffer Red is a ready to use PCR buffer, including all components for standard PCR applications. FEATURESAll in one buffer for highest convenienceDirect gel loading onto agarose gelsRed colour enables easy visualisation of pipetting and loadingDye front runs at 1000 - 2000 bp on DNA 0.5 - 1.5 % agarose DESCRIPTION5X PCR Buffer Red offers the same specification as our Taq-E buffer. It results in high yield of PCR products and minimises the need for optimisation of Mg2+ concentrations or the annealing temperatures. The included red dye and density reagent, eliminates the need for loading dye as well as the time consuming sample preparation before electrophoresis.