Can qPCR primers be used for PCR?

The primers that work on qPCR, can actually work on endpoind PCR. However, the rules for designing primers for qPCR are more restrictive. It depends on which detection method you are going to use (Taqman probes of SYBR Green Dye).

What primers are used in qPCR?

Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®).

Do you need primers for qPCR?

qPCR primer design is a critical step when setting up your qPCR or reverse transcription-qPCR assay (RT-qPCR). qPCR primers that anneal poorly or that anneal to more than one sequence during amplification can significantly impact the quality and reliability of your results.

What is the optimal length of a qPCR primer?

between 70 and 200 bp
Length: For typical cycling conditions, ideal amplicon size is between 70 and 200 bp. Longer amplicons can be designed, but cycling conditions should be adjusted to include longer extension times.

How are copies of EGFP detected in qPCR?

A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was ( i) highly reproducible, ( ii) detected as few as two copies, and ( iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains.

What are the primer and probe sequences for eGFP?

Primer and probe sequences for EGFP were from Jackson Laboratories (personal communication) and manufactured by Applied Biosystems: forward primer 5′-ccacatgaagcagcaggactt-3′, reverse primer 5′-ggtgcgctcctggacgta-3′ and probe 5′-6FAM-ttcaagtccgccatgcccgaa- TAMRA-3′. The final concentration of EGFP primers was 400 nM each, and the probe was 150 nM.

Can a sequence be used as a reverse primer?

Cheers! If you want to use the aforementioned sequences by Mr Munagala as forward and reverse primer, you must reverse complement the proposed sequence for reverse primer. Therefore, after checking the physicochemical properties, you can use the following sequences as forward and reverse primer:

How is real time PCR used for biolocalization?

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells.