Will the TaqMan probe length affect the fidelity of the qPCR assay?

Dear all, I am learning to design the qPCR assays recently. But I just don't know why my negative controls always give positive signal( I have changed my design twice). I checked the formation of...

09 October 2019 7,082 3 View

Effect of mRNA secondary structure on reverse transcription?

Dear Sir/ Madam, I am designing a primer for microRNA reverse transcription. But some microRNAs like hsp-mir-21-5p has a strong secondary structure. It forms a stable hairpin. Can anyone please...

08 September 2019 6,694 3 View

Effect of template secondary structure on the PCR?

Dear all, I am designing a pair of primers for PCR, but my target region has very severe secondary structure(eg. hairpin with 15-bp stem). Can anyone please tell me whether the secondary...

08 September 2019 6,799 6 View

Annealing temperature set-up for short primer of 11 bases?

Dear all, I am amplifying a target sequence of 65 bases, which is a cDNA sequence now. For some reasons, my PCR primer can only bind to the target sequence with 11 bases in the first PCR cycle....

09 October 2018 9,483 8 View

Incomplete quenching of molecular beacon?

Dear all, I am detecting mRNAs with hairpin molecular beacon. The 5' end of the hairpin oligo is modified with BHQ1 and the 3' end FAM. After I dissolved the hairpin in NF water, I dispensed one...

07 August 2018 424 5 View

Effects of non-ionic detergent on double strand DNA binding affinity?

The non-ionic detergent can be used as additives in the PCR to reduce the secondary structure, which might be helpful for the low yield PCR. But it may also increase non-specific amplification....

03 April 2018 4,930 2 View

Acrylamide hydrogel beads get cross-linked and form clusters?

I am making hydrogel beads with microfluidic droplet. The aqueous phase is a mixture of acrylamide, AA/bis-acrylamide, APS, and water. The oil phase is fluoroOil and fluoroSufactant with 0.4%...

11 December 2017 4,398 3 View

Can anyone please tell me if the sodium metaperiodate will degrade mRNA?

05 June 2017 7,648 2 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

Research on Supply Chain Management in Indian context?

Hi, I am planning to apply for the PhD degree in the Supply Chain Mgt. with specific area of "Cold Storage warehouses" during Pandemics and wars. Where lock downs and shut downs are frequent....

02 March 2021 285 2 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

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 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

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View

Can thermal cycler machines be problematic during PCR?

Hi, my question is about the heating of thermal cycler machines and I hope some of you had experienced a similar thing previously. There are two thermal cycler machines in the lab(BioRad) and for...

26 February 2021 4,777 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