How much concentration of primers is ideal to use for sensitivity?

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The primers that I have are amplifying lower concentration of DNA but not higher. Is this even possible? What can be done to fix this?

I am trying to do qPCR using some primers. The primers are specific for lower concentrations. However, I do not see any amplifications at higher concentrations. What could be the reason for...

06 July 2019 4,201 1 View

Why are most greenhouse studies having small population and sample sizes as compared to the ones described in statistical books?

I am a beginner in experimental design so maybe I am not understanding the basics. In a class that I took , for a given population and confidence level and interval, the recommended sample size is...

01 January 1970 936 0 View

What is the usual concentration of Diaporthe fungus using DNeasy mini kit protocol?

Hello Everyone, I am using DNeasy mini protocol to extract DNA from at least 7 days old culture in PDA. When I tried it, several times I am getting less than 10 ng/micro liter of DNA. It is...

01 January 1970 1,393 2 View

I am trying to extract DNA of fungus from plant tissue and amplify the fungal DNA. Any suggestions on the protocol?

Can anyone explain how it works. The fungus lives in the vascular bundles.

01 January 1970 7,572 2 View

The housekeeping gene stability analysis?

I am doing real time PCR , relative quantification for a fungus that infects soybean. What housekeeping genes do I use for this? Are these for the fungus itself? I did not find them for my fungus...

01 January 1970 938 1 View

Can I prepare standard curve from DNA extracted from pure culture of my fungus for absolute quantification?

I am working on a fungus and was wondering if I can extract DNA from the pure culture of the fungus and measure the concentration of DNA and do a serial dilution for absolute...

01 January 1970 6,554 4 View

What is the best protocol for qPCR of fungal tissue?

I am analyzing the process of colonization by a fungus on plant stem. I am trying to do real time PCR for this process. In the meantime I am looking for papers, I am finding different processes...

01 January 1970 9,337 4 View

Can we analyse the reference gene expression without software mentioned in literature like Normgne, REST etc?

I am trying to calculate the reference gene expression stability for my qPCR assay since nobody has worked in case of my soybean. I am working on relative quantification of a fungus inside soybean...

01 January 1970 562 0 View

Trying to replicate the research paper , but the thermal cycling conditions of the primers do not work. Why?

I am trying to replicate using a set of primers mentioned in a paper Quantification of Fusarium solani f. sp. glycines isolates i... The thermal cycling conditions mentioned are 95 degree...

01 January 1970 1,488 9 View

In PRIMER, is it possible to downweight certain taxa in multivariate community datasets to reflect uncertainty around identification??

I have a dataset with about 80 different species. As usual, some species are very easy to identify with certainty whereas others are more difficult, which means that I am less certain of my...

03 March 2021 8,066 4 View

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 View

How to calculate the annealing temperature of a primer pair?

I have to amplify a gene and my primers just reached. The Tm for Forward primer is 64.2, and that of reverse primer is 65.5. Can some one suggest how to get the best annealing temperature? Thanks...

01 March 2021 360 7 View

Anybody with an idea on how do design specific primers for detecting tomato resistance genes tm-1, TM-2 and Tm2^2?

I am trying to identify these 3 genes among some tomato cultivar collections and after aligning some sequences from NCBI, I couldn't find unique sequences to target for specific primers. There...

28 February 2021 606 3 View

What is the ideal efficiency for a qPCR?

hello everyone, I need to do standard curves for my qPCR, what is the ideal efficiency range? I tried a primer (Mglu2 receptor) that gave an efficiency of 90.2%. Is it accepted?

28 February 2021 1,254 3 View

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Calculating volume of DNA required from DNA concentration?

Hi I am a bit confused. They are asking me to find out the volume of DNA required in ul (a total of 30-100 ng for genomic DNA) from the DNA concentration in the nanodrop reading which was 404.8...

26 February 2021 5,029 2 View

Has anyone used the Illumina ITS1 primer sets?

Hi everyone, Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each...

25 February 2021 6,969 3 View

Genescan gives 3 peaks instead of one?

Hi all, I am doing a genescan analysis for a deleted exon in the patient sample. The amplified region is 215 bp. In the patient sample because of homozygous deletion, no peak is observed while in...

23 February 2021 5,645 1 View

QPCR reaction efficiency with odd results what does this indicate?

I am working on a CFX 96 machine to determine reaction efficiency of my gene of interest. After performing a serial dilution of my cDNA template, I was able to find great reaction efficiencies for...

23 February 2021 809 3 View

Ahmed Dhamad

I assume you want to detect a plasmid that has been introduced to a cell. Depending on your polymerase, you could choose the best primer concentrations. Check the instructions that is specific to your polymerase and see what the final concentration of primers that was recommended is. Good luck

Alina Avanesyan

You may also check the instructions for your PCR-mix, it should contain suggested concentration and proportion for primers, DNA template, etc. 10um seems very high, I usually worked with 4 or 2um, however I didn't work with plasmids. Best of luck!

Hassan Nima

agree with@ Ahmed Dhamad

regards

Ashraf Abbas

you should optmize the condition reaction ( DNA conc, DNA Quality PCR cycles Number