PCR

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3 Why is there inconsistency in the Ct value of real time PCR?
By Nyuk Kon · University Malaysia Sarawak

Jack Gallup · Iowa State University

A good representative mixture of appropriate DNA samples wherein your targets are likely to exist with good abundance would be the ideal stock to use ... [more]

A good representative mixture of appropriate DNA samples wherein your targets are likely to exist with good abundance would be the ideal stock to use for standard curve purposes. But, as we all know, part of that is a guessing game: i.e., which samples does one mix to get a good stock ? - is always the question - unless more is learned as to which samples are the best to use for this purpose over time. It appears that your second stock dilution series study may have been much less concentrated than your first attempt(s); thus the large increase in Poisson noise that Luciano speaks of. A fluctuation of 2 Cq units among technical replicates is pretty severe ... Other possible causes: an electronic pipet (on multi-dispense mode) that delivers the replicate repeat sample volume consistently differently than the first replicate sample volume delivery...

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3 Melting curve analysis software
By Antonio Galiana · FISABIO

Ghulam Dhabaan · University of Malaya

Try GeneEx, it can help. try the trail version. best http://genex.gene-quantification.info/

Try GeneEx, it can help. try the trail version. best http://genex.gene-quantification.info/

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3 RNA ladder
By A.K.M. Moyeenul Huq · National University of Malaysia

A.K.M. Moyeenul Huq · National University of Malaysia

Thank you guys for your suggestions.

Thank you guys for your suggestions.

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19 PCR purification for sequencing.
By Konstantin Kitaev · Ufa Scientific Center of the Russian Academy of Science

Koen Venken · Baylor College of Medicine

If the band is pure, you can directly purify with a PCR purification kit. If not, run on a gel, cut the right band out, and purify with a gel extracti... [more]

If the band is pure, you can directly purify with a PCR purification kit. If not, run on a gel, cut the right band out, and purify with a gel extraction kit. Any of the commercial kits work well (Qiagen, Zymo, ...)

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1 How do I achieve a smooth melt curve for my results?
By Huiyi ow · Temasek Polytechnic

Natalia Koretskaia · Moscow State University of Medicine and Dentistry

Is this HRM analysis of genotypes? Our Rotor-Gene3000 can not do it. But in the analysis of an gene expression (our Rotor - Gene 3000) the such is ob... [more]

Is this HRM analysis of genotypes? Our Rotor-Gene3000 can not do it. But in the analysis of an gene expression (our Rotor - Gene 3000) the such is observed when the incorrect begin calibration wizard.

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18 TaqMan no-RT controls showing amplification that is not from genomic DNA in real time quantitative PCR.
By Ammon Thompson · University of Texas at Austin

Josef Buttigieg · University of Regina

With primer design it is usually best if you can have it spanning 2 exons. Thus only transcribed info will show up in PCR (not genomic). now if that i... [more]

With primer design it is usually best if you can have it spanning 2 exons. Thus only transcribed info will show up in PCR (not genomic). now if that is the case and you are still getting a +ve, it could be that you have a reverse transcriptase contamination in one of your reagents. thus amplifying your RNA (false positive).

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27 I am going to design a real time PCR experiment, what things should I keep in mind?
By Himanshi Kapoor · University of Delhi

Nathaniel Mcgregor · Stellenbosch University

Dear Himanshi Firstly you need to decide whether you need to perform absolute or relative quantification. This will depend on your initial question ... [more]

Dear Himanshi Firstly you need to decide whether you need to perform absolute or relative quantification. This will depend on your initial question e.g determining HIV viral load within a sample (absolute) or assessing gene expression relative to a reference control (relative). You set-up will then also largely depend on the machine you have available to you. For examle Roche's LightCycler 480 I/ II will allow for you to practically set up everything on a single plate and perform the relative quantification for you! Another machine will require a little bit more prep work and additional reactions. The MIQE standards mentioned earlier are an absolute. They will make publication and quite frankly validation much easier!! They speak to selecting the correct reference gene for your tissue sample (there are websites that will aid in this) etc. It is also important to remember that if you're using a standard curve you need to include your standards, patients and controls all on a single plate in a single run in at least duplicate appearances with negative controls. For cDNA conversion based real-time a genomicDNA control is essential as well. If you're going to calculate via the 2 delta delta Ct method you need to ensure that you're PCR inefficiencies for each primer pair being compared is relate able - or you results will not be accurate. The are many other things but I find that those I've mentioned above are what most first-time experiment runners tend to slip up on. They're minor things but can be quite time- consuming and in some cases expensive to repeat once discovered. Hope this was helpful. Regards Nathaniel

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17 What PCR Thermal Cycler(s) would be recommended to replace an old MJ Tetrad PTC-225?
By Ian Herriott · University of Alaska Fairbanks

Raghavendra Kulkarni · SDM College of Medical Sciences & Hospital

Quanta Biotech is an excellent alternative. I have been using it for last 5 years. Very reliable performance. The software is user friendly.

Quanta Biotech is an excellent alternative. I have been using it for last 5 years. Very reliable performance. The software is user friendly.

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7 Effects of Tags on Over Expression?
By Daniel Tagoe · University of Cape Coast

Daniel Cohen · University of Pennsylvania

Kalpesh is right, but you may simply have to take this risk since you are focusing on predicted proteins. These overexpression experiments may be sugg... [more]

Kalpesh is right, but you may simply have to take this risk since you are focusing on predicted proteins. These overexpression experiments may be suggestive of subcellular compartmentalization or function, but will by no means will prove them.

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5 Non-conventional PCR protocol: 2 annealing temperatures per cycle.
By Béatrice Gaume · Université des Antilles et de la Guyane

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10 PCR product with smear
By Manuela Marescotti · The University of Edinburgh

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9 How to successfully amplify a 6kb fragment of DNA?
By Yiren Xiao · Chinese Academy of Sciences

Subhradip Karmakar · Yale University

You results seems to typical case of vector self ligation . First make sure that the RE are working fine (run gel and check) . Also you need to minim... [more]

You results seems to typical case of vector self ligation . First make sure that the RE are working fine (run gel and check) . Also you need to minimize self-ligated vector in your transformation, for that treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should improve the percentage of colonies with insert. Lastly make sure that the PCR worked! since its pretty long target, I am skeptical about the eff of the polymerase

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7 What problems could occur if the concentration of DNA in a PCR reaction is more than 200 ng?
By Sahil Patel · Directorate of Groundnut Research

Jack Gallup · Iowa State University

200 ng in a 100 uL qPCReaction? 50 uL, 25 uL, 20 uL or 10 uL reaction? Here you see you would have 2 ng/uL (which is OK), 4 ng/uL, 8 ng/uL, 10 ng/uL ... [more]

200 ng in a 100 uL qPCReaction? 50 uL, 25 uL, 20 uL or 10 uL reaction? Here you see you would have 2 ng/uL (which is OK), 4 ng/uL, 8 ng/uL, 10 ng/uL and 20 ng/uL, respectively. I would suggest doing a serial 1:2 dilution study (20 or 30 points) of your DNA and see how the reactions behave. If you have too much target in the beginning reactions, you will see template inhibition of the reaction(s) (Cq values higher at first, then they will get lower, and then start responding to dilution correctly).

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15 Steps involved in designing PCR/qPCR primers?
By Ying Hui Ong · University of Malaya

Chris Pedigo · University of Miami Miller School of Medicine

There are quite a few databases that you can use that have the primers already designed! I would still primerblast them to double check them, but on ... [more]

There are quite a few databases that you can use that have the primers already designed! I would still primerblast them to double check them, but on the harvard site some of them are already tested so that saves you some trouble. What i normally do is to order 2-3 sets per gene from this site, and test them on some normal untreated cDNA and run melting curves and an agarose gel to ensure I only get one product. I just think this is easier than the above suggestions. Good luck! http://pga.mgh.harvard.edu/primerbank/ http://primerdepot.nci.nih.gov/

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12 Problems cloning a 300 nucleotide fragment with XhoI and EcoRI restriction sites in pHannibal vector.
By Mohammad Jafari · Ferdowsi University Of Mashhad

Jorge Garcia · Centro de Investigación Científica de Yucatán

Hi Mohammad, I was having similar problems with pKANNIBAL so, I cloned my PCR product in pGEM and I sent to be sequenced, in the results of sequensati... [more]

Hi Mohammad, I was having similar problems with pKANNIBAL so, I cloned my PCR product in pGEM and I sent to be sequenced, in the results of sequensation I found that my PCR product realy haven't the sequence for the restriction enzimes (despite it is in my primers sequence), now I'm still trying to solve this problem.

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9 Sample volume in real time PCR
By Chandan Kadur Nagaraju · Jawaharlal Nehru Centre for Advanced Scientific Research

Joanne Hassan · Kenya Medical Research Institute

hy chandan, we use Applied Biosystems 7500 standard system for our PCR, when we forget to change during the set up we change after the run and it does... [more]

hy chandan, we use Applied Biosystems 7500 standard system for our PCR, when we forget to change during the set up we change after the run and it doesnt affect the results

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9 Why is there dimer formation in PCR?
By Bhawik Jain · Advanced Centre for Treatment, Research and Education in Cancer

Tejas Bosamia · Junagadh Agricultural University

Try different primers set for target.

Try different primers set for target.

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14 Is it possible to have amplification with primer, its melting temperature (40) and the annealing temperature 59C?
By Youssuf Gherbawy · South Valley University

Tejas Bosamia · Junagadh Agricultural University

GC content of the product might be to high that it was not able to generate further products, so DMSO, BSA, Glycerol may be useful in this case. anot... [more]

GC content of the product might be to high that it was not able to generate further products, so DMSO, BSA, Glycerol may be useful in this case. another possibility, primer have stem loop structure which prevent binding of primer to template.

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6 Which primers can amplify the largest range of algae?
By Nataliya Sachovska · Wayne State University

Fernando Martínez-Alberola · University of Valencia

May be this primer pair is of your interest: CAMPO LÓPEZ, Eva María del; CASANO MAZZA, Leonardo M.; HOYO PÉREZ, Alicia del; ROYO , Carolina; ÁLVAR... [more]

May be this primer pair is of your interest: CAMPO LÓPEZ, Eva María del; CASANO MAZZA, Leonardo M.; HOYO PÉREZ, Alicia del; ROYO , Carolina; ÁLVAREZ CUBERO, Raquel; BARRENO , Eva. "A single primer pair gives a specific ortholog amplicon in a wide range of Cyanobacteria and plastid-bearing organisms: applicability in inventory of species and phylogenetic analysis" (ISSN:1055-7903). Molecular Phylogenetics and Evolution. 2010, vol 57, p. 1323-1328

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10 Any suggestions for RNA extraction of tissues with high chitin content?
By Jose Manuel Estivalis · Fundação Oswaldo Cruz

Raffaele Dall'Olio · CRA Agricultural Research Council

another risky Ad-post: Qiagen RNeasy kit already have the double column (the first one to remove debris and avoid column clumping)!

another risky Ad-post: Qiagen RNeasy kit already have the double column (the first one to remove debris and avoid column clumping)!

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24 How can I minimise the PCR inhibition when using bone samples?
By Hannah Tanner · Public Health England

Daniela Polverino · National University of La Plata

Hi Hannah, I agree with you respecto to PVP-TE, if it´s for diagnostic is no useful. Perhaps you use Expand Long DNA polimerase for your PCRs, this p... [more]

Hi Hannah, I agree with you respecto to PVP-TE, if it´s for diagnostic is no useful. Perhaps you use Expand Long DNA polimerase for your PCRs, this polimerase woks very well with inhibotories. good luck.

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93 How to amplify a specific region of genomic DNA via PCR?
By Forest Ray · Columbia University

Kimberly Dyer · National Institutes of Health

I have had good success amplifying difficult targets using nested PCR reactions.

I have had good success amplifying difficult targets using nested PCR reactions.

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3 Does anyone know some protocol for RNA amplification?
By Neus Bota · University Pompeu Fabra

Neus Bota · University Pompeu Fabra

Hi and thx for your answers! The problem I have is that I extract RNA from a small part of living organism, and it's very difficult to get samples, so... [more]

Hi and thx for your answers! The problem I have is that I extract RNA from a small part of living organism, and it's very difficult to get samples, so my total amount is very low. I'll try to do RT PCR and see if I get enought cDNA for the real times. And the kit you've shown me looks pretty good, I'll talk with my supervisor about that! Thanks a lot :)!

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3 Isolating DNA from fishes.
By Owais Wani · Dr. Harisingh Gour University

Adrian Blake · Bournemouth University

Fin tissue is a good way to take DNA if you have Ethanol to store the fin in. Take a good sized chunk of one of the ends of one of the lower fins (it ... [more]

Fin tissue is a good way to take DNA if you have Ethanol to store the fin in. Take a good sized chunk of one of the ends of one of the lower fins (it grows back fairly well). Otherwise, I use scales, which can be removed with tweezers and then kept in small envelopes. The scales dry and encase mucous, meaning that the DNA is preserved. I have just been extracting DNA from scales that have been kept in envelopes in a drawer for over 10 years. Scales give less DNA, but it is easier to store and is less intrusive. There are a number of online protocols specific for extracting from scales, but I find it helps to just use twice the normal amount of Proteinase K as you would use for a normal tissue extraction. Both of the above can be used whilst causing minimal distress for the fish (though I suggest anaesthetic for the fin clipping). If you have a dead fish, then go for the muscle, 20mg should be enough, about the size of a fingernail clipping.

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3 How to reduce nonspecific hybridizations on a real time PCR assay?
By Antonio Galiana · FISABIO

Antonio Galiana · FISABIO

Thank you very much for your help

Thank you very much for your help

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100 His tagged protein not binding to beads
By Fabio Da Silva · McGill University

Swati Ghosh · National Institutes of Health

Please use hepes buffer (ph7.4)

Please use hepes buffer (ph7.4)

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5 What should the concentration of primers be when I am doing multiplex PCR?
By Sahil Patel · Directorate of Groundnut Research

Suma Paramesh · Ashoka Trust for Research in Ecology and the Environment

3pmole forwardprimer and 1.5 pmole each of reverse primer should be fine. I usually use 3pmole concentration for multiplex PCR

3pmole forwardprimer and 1.5 pmole each of reverse primer should be fine. I usually use 3pmole concentration for multiplex PCR

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