#Qcast web not playing how to#

The plasmid pair had the same size (3306 bps) and could be easily manipulated with identical copy numbers. They were used to establish quantification standards. Thirdly, allele-specific (AS) primers and a FAM-containing TaqMan probe (Bio-Rad, USA) were used for JAK2V617F detection, whereas primers for the control gene were mixed with a HEX-containing probe to quantify JAK2 exon 21 ( Figure 1C), and the calculation of mutant AB was determined by the ΔCt method. The designs allowed us to perform duplex PCR (including mutant-specific assay and copy-number-normalizing control assay) in one tube and quantify two PCR products concurrently, which also helped minimize sampling bias and decrease the consumption of genomic DNA. The latter could be a major but unexpected benefit when some rare samples of limited quantity are examined. Lastly, but most importantly, we designed an oligonucleotide (ON) WT template blocker containing a di-deoxycytidine at its 3′ end (3′-ddCTP). It competed with the mutant allele-specific primer and preferentially annealed to WT templates with high affinity, which impeded their non-specific amplification. With these refinements, we designated our novel method as quantitative competitive allele-specific TaqMan Duplex PCR (qCAST-Duplex PCR) assay.

The sequences and working concentrations of all primers, probes and the blocker are listed in Online Supplementary Table S1. Other relevant information is also available in the Online Supplementary Appendix. The reference sequence, plasmid construction, and validation of the accuracy of artificial plasmid mixtures as quantification standards. (A) Physical map of JAK2 exon 14-22, including the mutation position on exon 14 and the 264-bp reference control region on exon 21. The asterisk indicates the JAK2V617F mutation position (cDNA 1849 G>T).

#Qcast web not playing how to#

You can find out how to avoid them below.The arrow heads show the primers which amplify the reference control sequence. These cookies and other technologies capture data like your IP address, when you viewed the page or email, what device you were using and where you were. We use various advertising partners, including Amazon, Facebook, and Google. These cookies are used to track your activity on the BenQ website and other websites across the Internet, help measure the effectiveness of our advertising campaign and deliver advertisements that are more relevant to you and your interests. See list of performance and advertising cookies To opt-out of Hotjar collecting data, you can disable tracking completely by following link：. To opt-out of SessionCam collecting data, you can disable tracking completely by following link：. To opt out of certain ads provided by Google you can use any of the methods set forth here or using the Google Analytics opt out browser add-on here. You can control the information provided to Google, SessionCam and Hotjar. If you want to opt-out of advertising cookies, you have to turn-off performance cookies. We also use Google Analytics, SessionCam and Hotjar to track activity and performance on the BenQ website. These cookies help to improve the performance of BenQ. Performance cookies and advertising cookies