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Standard PCR

PCR Optimization: Reaction Conditions and Components new protocol
Consideration for components of the PCR optimization: Primer Design: Oligonucleotides used for priming the PCR should be at least 16 nucleotides and preferably 18-25 nucleotides in length. They should contain 40% -60% G+C. Avoid sequences which would produ ...

Standard PCR Protocol protocol
From Molecular Biology Techniques Manual. Protocol written by Ed Rybicki , January 1994, February 2001. Content Recommended Reagent Concentrations * Recommended Reaction Conditions * Initial Conditions * Temperature Cycling * "Hot Start" PCR * ...

PCR trouble shooting new hot recommended troubleshooting
A nice troubleshooting guide on PCR reaction including a detailed PCR troubleshooting flowchart and many useful tips and problem-solving suggestions. (Department of Biology, University of Oslo) Some possible reasons on an unsuccessful PCR: A. Pilot error ...

PCR smearing trouble shooting troubleshooting
We have been getting a lot of smearing in our PCR reactions. Sometimes we get a discreet band, but most of the time its a smear. We get this with different primer pairs so it dosent't appear to be confined to one pair. The reactions are all set up on ...

dNTP problem and magnesium Mg concentration in PCR reaction troubleshooting
I have been trying to get a PCR started for 1 month, no results. I modified everything, especially annealing temperatures and DNA concentration. I took the template and primers to another lab, and boom, the reaction worked in 4 out of 4 samples. I came back an ...

Removal of genomic DNA contamination in RT-PCR troubleshooting
I have just begun RT-PCR experiments. They are going very well so far with beautiful amplification as show on agarose gel. However, when I run the no-RT control I have a very faint band indicating that I have not fully removed the DNA from my RNA. I m ...

PCR contamination: amplification in negative control new troubleshooting
PCR product band is observed in agarose gel electrophoresis from a negative control. Solution: 1. Work in a pre-PCR workspace ( i.e. PCR station with UV light) to avoid contamination from previously amplified DNA 2. Put on a fresh pair of gloves when be ...

High molecular weight PCR artefact troubleshooting
A student in our lab is reproducibly seeing a high M.Wt band (it only enters the gel a mm or so after an hours electrophoresis in 1% agarose) after PCR of genomic DNA. We think we've ruled out bacterial contamination (-ive Taq control does not show th ...

Taq inactivation troubleshooting
I am going to directly use the PCR mixture for restriction digestion, but I fear the remaining Taq in the PCR mixture will fill in the 5' overhang generated by restriction endonucleases. Is my worry necessary? Can Taq be inactivated by heating at ...

Unknow Band from PCR product troubleshooting
I have run my PCR products on a 1% agarose gel with TAE buffer. However, I found a strong band migrating towards the negative electrode after EBr stain the gel. I still saw my product at the positive end that is apparently very normal. ... ... ...

PCR trouble shooting, help, suggestions troubleshooting
If your PCR amplification somehow performs unexpectedly, it is usually caused by one of the listed possible errors - ranked by frequency. You may try checking if the problem is repeatable, see trouble shooting flowchart. Be patient and follow a standard ...

Paraffin embedded tissue PCR troubleshooting
PCR Methods and Applications, Vol 1, 46-50 -------------------------------------------------------------------------------- ARTICLES PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective ...

Standard PCR Troubleshooting and Optimization new troubleshooting
A general guideline for PCR optimization including reaction conditions and components (from Applied biosystems). Some considerations for standard PCR: 1. Sample Volume and Reaction Tubes 2. Template DNA or RNA 3. Primers 4. Deoxynucleoside Triphospha ...

PCR MasterMix Calculator software
Online tool to quickly calculate the amount of components needed to create your PCR MasterMix. (Sigma PCR tools) ...