Polymerase Chain Reaction Platform

• Vivek Kumar Vaishnav

  Taq DNA plymerase purity test?

  I am working with plant genomes. Recently i ordered units of Taq DNA polymerase, and results are not quite good. how can i test whether the supplied Taqs are pure or it has been contaminated?

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  • 
    Ajay Saini replied

    Hi Vivek Colud you please elaborate what type of contaminations you are expecting?....or you want to rule out...in the commercial Taq polymerase preparations..

    6 hours ago
• Bassethoundt Bassethoundt

  New real time PCR Machine - which one?

  For our lab we are considering the purchase of a new Real Time PCR Machine. After gathering some information about what the market of Real Time PCR machines offers, we’re not really sure whichFor our lab we are considering the purchase of a new Real Time PCR Machine. After gathering some information about what the market of Real Time PCR machines offers, we’re not really sure which machine might be the best choice for us. Basically we want to improve our processes in our projects and get better and faster results of our researches. I would highly appreciate if you can give me your opinion of your experiences with different Real Time PCRs in general, also in consideration of price/performance. That really would help us in our choice. Thanks for your input!

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    Sanket Joshi replied

    I would advice to go for Biorad ddPCR. recently launched .... droplet digital PCR. We recently purchased it (ABI is also good), not yet installed.

    6 hours ago
• Lifang du

  qRT-PCR Analysis

  I want to quantify the DNA copies of HSV2 in cervicovaginal lavage (CVL) specimens, using the gene extracted from the virus as the standard. Ten-fold serial dilutions were prepared with the standard.I want to quantify the DNA copies of HSV2 in cervicovaginal lavage (CVL) specimens, using the gene extracted from the virus as the standard. Ten-fold serial dilutions were prepared with the standard. But when the copies were lower than 1000, the dot would deviate from the standard curve. How can I solve this problem?

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    Lifang du replied

    I had used the plasmid as the standard and then found it was amplified more efficiently than the genomic DNA. So the genomic DNA was used as the standard.

    3 days ago
• Agnieszka Paulina Kijewska

  High Resolution Melting

  Did you try to clear out some DNA from a bit degradated samples? I am thinking about HRM and I can't decide if I should clean out dNTPs first or just make a PCR and try HRM after using amplicons.

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    Agnieszka Paulina Kijewska replied

    Thanks Gustavo. Yesterday I used DNA purifying Kit to clean samples. I'll check them today. Contamination is excluded, rest of SNP is working well...

    3 days ago
• Lucas Low Van Lun

  Determination of heterozygous/homozygous based on sequencing method

  Currently I am working on point mutation detection based on direct sequencing method. I found that certain mutation site exhibited two peaks in the sequences. Can I clarify that this is heterozygous?Currently I am working on point mutation detection based on direct sequencing method. I found that certain mutation site exhibited two peaks in the sequences. Can I clarify that this is heterozygous? Would it be reliable for the determination of heterozygous/homozygous based on this sequencing method? Thanks.

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    Lucas Low Van Lun replied

    Thank you very much for the comments and suggestions. Thank you. Appreciated.

    4 days ago
• Omar J BenMarzouk-Hidalgo

  Real-time immuno-PCR

  I am trying to set up a new rt-IPCR assay in my lab in order to find out different amount of mature and inmature proteins for knowing virus activity and infectivity. I have come along differentI am trying to set up a new rt-IPCR assay in my lab in order to find out different amount of mature and inmature proteins for knowing virus activity and infectivity. I have come along different papers advising different ways of obtaining the DNA conjugated antibodies, some use biotin-streptavidin protocols, while others use magnetic or even gold beads for the coating antibodies. I would like to know which one you recommend me to start with. In addition, should I use direct DNA-covalent binded antibodies or streptavidin intermediators? Thanks!

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    Muhammad Sajid replied

    I think you have to choose any of them based upon their intreaction with proteins of your interst. Also it depends upon ability and nature of your detector. Will you use some enzymatic reactionI think you have to choose any of them based upon their intreaction with proteins of your interst. Also it depends upon ability and nature of your detector. Will you use some enzymatic reaction between captured protien and your substrate? Please give a brief sketch of your experimental procedure, then we can discuss more about it.

    4 days ago
• Xiaoyu Zhao

  Optimization of Real Time PCR

  How can I optimize my real time pcr conditions? standard curve of Real time pcr (absolute quantification) : Low copies of standard plasmids (10-100copies) always have lower CT value.

  

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    Perwez Alam replied

    As per depicted from your Std curve you may try two more things, 1. If its OK, you can start from 100 instead of 10 because Ct values for 10, 100 and 1000 are not according to concentrations youAs per depicted from your Std curve you may try two more things, 1. If its OK, you can start from 100 instead of 10 because Ct values for 10, 100 and 1000 are not according to concentrations you used (but nearly similar), it might be due to very low concentration of sample (10), for same you put a blank and compare its Ct value with lower one. 2. you should recheck the concentration of your dilutions particularly for 1000 and 10000, are they actually, because due to handling error concentration may vary. note that even the real time PCR is a sensitive tool but it works very straight according to concentration. Good Luck

    5 days ago
• Ersin Karataş

  May I use 10X Dream Taq Green Buffer as a loading dye?

  I have too much 10X Dream Taq Green Buffer and not too much Loading Dye. So I want to use 10X Dream Taq Green Buffer as Loading dye. Is that possible? Is there anyone who use it before ?

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    David Jarrell replied

    You'd need to figure out the concentration... look at how much is used as a proportion of a PCR reaction... that will give you how you might need to use if using it solely as a loading buffer (I'mYou'd need to figure out the concentration... look at how much is used as a proportion of a PCR reaction... that will give you how you might need to use if using it solely as a loading buffer (I'm assuming you are not using it for PCR because the green dye can interfere with downstream applications such as sequencing).

    5 days ago
• Lavender Agutu

  I have PCR products that need sequencing. However the DNA concentration is too little for possible sequencing. How do I concentrate the DNA?

  I have run the PCR process several times getting the same light band results. I have also tried using more of the DNA template with unsuccessful results. If however anyone knows something about LAMPI have run the PCR process several times getting the same light band results. I have also tried using more of the DNA template with unsuccessful results. If however anyone knows something about LAMP procedure kindly let me know if its possible to purify from LAMP products

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    Shan Goh replied

    MinElute by Qiagen works well for purification and concentration of amplicons less than 4kb. Try to pool as much of your PCR product as possible and run it through MinElute. The elution volume isMinElute by Qiagen works well for purification and concentration of amplicons less than 4kb. Try to pool as much of your PCR product as possible and run it through MinElute. The elution volume is typically 10ul.

    5 days ago
• Yung An Chua

  I got band smearing in my agarose gel electrophoresis. The smear was quite strange where it stopped exactly at a specific bp.

  My PCR template was a PCR amplicon amplified from human DNA (nested PCR) which has been diluted 200 times. Taq buffer conc. = 1X, MgCl2 conc. = 2.5 mM, dNTP = 0.2 mM, Taq polymerase = 0.5 U. Four setMy PCR template was a PCR amplicon amplified from human DNA (nested PCR) which has been diluted 200 times. Taq buffer conc. = 1X, MgCl2 conc. = 2.5 mM, dNTP = 0.2 mM, Taq polymerase = 0.5 U. Four set of primers were used for a single reaction (multiplex PCR). As shown in the picture, the band with a green arrow was my intended band (960 bp). The smear was stopped exactly at 700 bp and the smear appeared as a 'ladder shape' with discrete bands. Adjusting the annealing temperature did not solve the smearing problem where every time I managed to make the smear gone, the intended band was also disappeared. What other parameter that I still can attempt to optimised? My DNA template is a GC-rich template. Do i have to increase the melting temperature? Thank you.

  

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• Sonalika Kar

  Thanx for answering the previous question.now plz anybody can tell me what qualities are required to be a good primer?

  How can we check the specificity of a good primer?

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    Sonalika Kar replied

    Thanks everyone..!!!!

    6 days ago
• Shambhavi Shubham

  No band in PCR after reverse transcription

  My end goal of the experiment is to do a qPCR of the RNA samples. I isolated RNA but didnt get very good yields and also the 230 peak was very high. Still, I went ahead with reverse transcription andMy end goal of the experiment is to do a qPCR of the RNA samples. I isolated RNA but didnt get very good yields and also the 230 peak was very high. Still, I went ahead with reverse transcription and PCR. My positive control samples also didn't work. Any suggestions as to what might have gone wrong. Also, I am new to this area, so could anyone explain the reasoining behind doing a standard curve in qPCR and what sample should be used for making the standard curve. I would appreciate any suggestions. Thanks, Shambhavi

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    Vanessa Tan replied

    like mentioned before, you probably have a contaminant in your RNA that is hindering the PCR reaction such as residual ethanol. if you wanted to, you could probably do a ethanol precipitation, but ilike mentioned before, you probably have a contaminant in your RNA that is hindering the PCR reaction such as residual ethanol. if you wanted to, you could probably do a ethanol precipitation, but i would recommend starting from scratch again, and making sure that you remove ALL the ethanol in the washing step. you need a standard curve to be able to determine the primer efficiency. it tells you how efficient a set of primers work, and is important when you want to compare between primers (eg when you use the 2 delta delta ct method to compare relative quantities of two genes of interest). if the primer for gene A has an amplification efficiency of 99%, while the other primer for gene B has an efficiency of 70%, then it is not reliable to compare between the two primers simply because the products are being produced at different rates. Like Krzysztof mentioned, you dont have to do this in every experiment. you only need to do it for every new primer pair to determine the efficiency. from them on you're okay! good luck!

    6 days ago
• So-Jin Kim

  Primer for mouse MKP-1 DNA

  It is the first time I order primer for specific DNA. I want to make a primer (reverse PCR) for mouse MKP-1 DNA and I don't know how can I make it! Can I have any information needed to make primerIt is the first time I order primer for specific DNA. I want to make a primer (reverse PCR) for mouse MKP-1 DNA and I don't know how can I make it! Can I have any information needed to make primer sequence? or any site which has explanation of reverse PCR primer? I think it's really great that who have been made mouse MKP-1 primer for reverse PCR (not real-time PCR). Thank you.

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    Kyoung-Ho Pyo replied

    Sound good~ ^^ have a nice experiment

    7 days ago
• Vennobaahshini Venu

  How to perform positive clone analysis using PCR?

  What combination of vector specific primer and insert specific primer should be used in the PCR reaction mixture? (eg ; vector specific primers forward or reverse with insert specific primers forwardWhat combination of vector specific primer and insert specific primer should be used in the PCR reaction mixture? (eg ; vector specific primers forward or reverse with insert specific primers forward or reverse) and what the size of the band i should expect? (insert size band or insert plus with vector size band)

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    Vennobaahshini Venu replied

    Thanks for the useful info.

    7 days ago