12 Questions 34 Answers 0 Followers
Questions related from O. Marte
We have a PCR assay whose product is later sent for simple DNA sequencing. Because we were restricted with our primer design (high A-T content) we were only able to design 1 set to use for both...
05 May 2018 5,450 14 View
Hello, I am designing a Taqman assay and have used ABI's Primer Express software to design the primers and probe. I've also had a bioinformatician check that the sequences are in conserved areas...
03 March 2017 8,803 2 View
There seems to be salt in my RNA after extraction. I proceeded to perform a cleanup step and while the quality (260/280) improved, the 260/230 remains low. I do not have more sample to...
05 May 2016 5,490 6 View
I am comparing 2 RT-PCR kits by using the same samples, looking at 2 targets. The top gel is one target, size 101bp. Lanes 1-4 are with one kit, where 1-3 are positive samples and 4 is NTC. Lanes...
01 January 2016 1,533 7 View
I extracted RNA from a positive and 2 negative samples. I then synthesized cDNA and performed RT-PCR. I inspected the amplification plot and melt curves for these. The positive sample came up on...
09 September 2015 7,601 15 View
I am validating my primer which will be used for relative quantification by doing a serial dilution. For 2 of my dilutions I have an outlier that is affecting my R2. I know an acceptable R2 is...
04 April 2015 9,750 6 View
I am trying to find the efficiency of my primers by doing a serial dilution of my sample, but the last 1 or 2 dilution points fail to amplify (or my triplicate points don't fall in the same Ct...
03 March 2015 7,628 4 View
The SYBR Green kit has the extension at 72C for 30 seconds. Should the time change if I decrease the temp?
03 March 2015 569 10 View
I am getting 2 different sequences, the Predicted RNA/mRNA Sequence (introns spliced out) and the Genomic Sequence (with introns). This gene has exons and introns and I want to design my primers...
03 March 2015 1,212 13 View
I need to have a final concentration of 500ng of cDNA in a 25ul reaction. Right now I have 950ng/ul. what is the math involved? What is a good cDNA final concentration to have for qRT-PCR? The kit...
03 March 2015 3,275 0 View
I will be using the primers for gene expression using RNA samples, but can I validate them first using gDNA or does it have to be with RNA, or both?
02 February 2015 3,996 16 View
I need to run a gel before sequencing it. What percent gel should I run it on, and what size ladder is adequate?
01 January 2015 4,948 2 View
