Navigation Links
Using Gradient PCR to determine the optimum annealing temperature.

Problems with the amplification of a specific DNA fragment using the PCR* are an everyday occurrence in the lab. For example, unspecific secondary bands may form after the PCR reaction, which hinder, or even prevent, further analyses (cycle sequencing, mutation detection, etc.) or an unequivocal assessment of the PCR result. In such cases, PCR conditions must be optimized. This is normally achieved by titrating the magnesium-, template-, primer-, dNTP- and Taq polymerase concentration, Hot Start PCR, Touch-down PCR, adding detergents, reducing the PCR cycles or by gradually increasing the annealing temperature. Fig. 1: Experimental determinati on of optimal annealing temperature: The calculated primer annealing temperature was 56.5C, the actual annealing temperature is 63.5C. The ribosomal spacer region of mycoplasms from H9 cell cultures was amplified. Using the gradient function of the universal block, a gradient of 53 to 67C was set. The following test parameters were selected: denaturation 10 s, annealing 15 s, elongation 20 s, amount of Taq-polymerase 0.75 units; duration of entire experiment: <1 h. Fig. 2: Amplification shown in fig. 1 performed under optimized temperature conditions. In this experiment, the universal block was set to a uniform temperature in the annealing phase. The outstanding temperature homogeneity of the block ensures reproducible PCR results. The selection of the annealing temperature is possibly the most critical component for optimizing the spec ificity of a PCR reaction. In most cases, this temperature must be empirically tested. The PCR is normally started at 5C below the calculated temperature of the primer melting point (Tm). However, the possible formation of unspecific secondary bands shows that the optimum temperature is often much higher than the calculated temperature (>12C).

Further PCR reactions with gradually increasing temperatures are required until the most stringent conditions have been found. When a standard PCR cycler is used, this method is the most time-intensive optimization strategy.

The Mastercycler gradient now enables rapid testing of the optimum temperature conditions on one block and in one experiment. During the PCR, a temperature gradient, which can be programmed between 1C and 20C, is built up across the thermoblock. This allows the most stringent parameters for every primer set to be calculated with the aid of only one single PCR reaction.

If the gradient function is not used, the block offers precise homogeneity, thus ensuring first-class reproducible results.

Example for Gradient PCR Program
'"/>

Source:


Page: All 1 2

Related biology technology :

1. Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library
2. Oil-Free PCR Using the Hot Top Assembly
3. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
4. Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
5. Using XL1-Red Mutator Strain to Generate Esterase Variants
6. Detect Released Oligosaccharides Using the Eagle Eye II Still Video Imaging System
7. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
8. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
9. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
10. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
11. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/9/2017)... ... October 09, 2017 , ... The award-winning American Farmer ... first quarter 2018. American Farmer airs Tuesdays at 8:30aET on RFD-TV. , With ... with the challenge of how to continue to feed a growing nation. At the ...
(Date:10/9/2017)... Antonio, Texas (PRWEB) , ... ... ... new study published on October 5, 2017, in the medical journal, Epilepsia, ... equivalence with the gold standard, video EEG, in detecting generalized tonic-clonic seizures ...
(Date:10/7/2017)... Mass. , Oct. 6, 2017  The ... work of three scientists, Jacques Dubochet, Joachim ... breakthrough developments in cryo-electron microscopy (cryo-EM) ... technology within the structural biology community. The winners ... Scientists can now routinely produce highly resolved, three-dimensional ...
(Date:10/5/2017)... LINDA, CA (PRWEB) , ... October 05, 2017 , ... ... innovators, engineers, and scientists from around the world, is giving back to cancer research ... sold in October. , Now through October 31, shoppers can use promo code ...
Breaking Biology Technology:
(Date:4/24/2017)... April 24, 2017 Janice Kephart ... with  Identity Strategy Partners, LLP (IdSP) , today ... without President Trump,s March 6, 2017 Executive ... , refugee vetting can be instilled with greater confidence, ... now, all refugee applications are suspended by until ...
(Date:4/18/2017)... , April 18, 2017  Socionext Inc., a global expert ... a media edge server, the M820, which features the company,s hybrid ... software provided by Tera Probe, Inc., will be showcased during the ... the NAB show at the Las Vegas ... ...
(Date:4/13/2017)... 13, 2017 UBM,s Advanced Design and Manufacturing ... feature emerging and evolving technology through its 3D Printing ... run alongside the expo portion of the event and ... demonstrations focused on trending topics within 3D printing and ... manufacturing event will take place June 13-15, 2017 at the ...
Breaking Biology News(10 mins):