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 I have asks for no more than 500ng.
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...
04 May 2018 5,493 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...
02 March 2017 8,827 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...
04 May 2016 5,536 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...
31 December 2015 1,566 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...
08 September 2015 7,632 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...
03 April 2015 9,829 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...
02 March 2015 7,665 4 View
The SYBR Green kit has the extension at 72C for 30 seconds. Should the time change if I decrease the temp?
02 March 2015 598 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...
02 March 2015 1,248 13 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?
01 February 2015 4,040 16 View
I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...
08 August 2024 4,645 2 View
After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...
08 August 2024 1,668 3 View
Hello, I suspect that a molecule I am using is causing bacteria to experience osmotic shock. What chemical properties should I look for to compare this capability with those of other substances?...
06 August 2024 977 1 View
i have sorted anti-NP specific plasma cells from bone marrow of C57BL/6 mice at certain times after immunization with variable counts and isolated total RNA using TRIZOL method for RT-PCR using...
05 August 2024 8,835 1 View
I have been attempting to extract DNA from Bacterial, Fungal and Yeast banked samples (>1e7 cells) using Prepman Ultra reagent and I seem to be struggling to obtain a sequence. Although the...
01 August 2024 2,079 0 View
Hello everyone, I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for...
31 July 2024 2,406 6 View
Can anyone with an MJ research / BioRad PCR machine from ~2010 or earlier tell me the external measurements (LxWxH) of the removable standard PCR alpha modules that can be removed from a PTC200 or...
30 July 2024 2,867 0 View
Hello all, I have been trying to follow a 2-stage PCR protocol used to amplify barcodes of a large yeast library, as per Nyugen et al. (2022) -...
30 July 2024 841 2 View
I need to install this program
25 July 2024 4,756 0 View
Hello, We found three packages of Illustra™ MicroSpin™ G-25 columns in the cabinet of an unused lab. They are very old but have never been opened. I have never used this kit before, and I couldn't...
25 July 2024 4,927 3 View