In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short... | Contact experts in PCR to get answers
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Questions related to PCR
Hi RG community! I'm writing this topic because one of my friends told me he has been experiencing evaporation issues with the Applied Biosystems' 9700, Veriti and ProFlex Thermal cyclers he has...
15 March 2021 8,690 10 View
Any details regarding what you should a should not vortex would be greatly appreciated!
11 March 2021 8,373 6 View
I have recently started working on gene cloning I want to digest my PCR product and my plasmid with BamH1 and Nde1 but I am not able to digest them using 1 unit of enzyme for 10 micro gram of DNA....
11 March 2021 4,718 7 View
I am attempting to optimize my RT-qPCR to run on Taqman Fast Virus Chemistry. My efficiency when running a standard curve is only about 80%. The amplicon produced is only approx 100bp and I get...
10 March 2021 7,671 2 View
We do not have a scale to monitor the LCO2 tank weight. Is there any other way to determine when 50% of the net LCO2 has been used? If useful, I am using it to dry bacterial biofilms on glass...
10 March 2021 1,315 0 View
Dear all I hope you are having a good time. I am trying to ligate a PCR product of ~1500bp into a 6.5kbp vector (6.023kbp upon digestion). Somehow, I am not able to get colonies at all ! The...
08 March 2021 3,631 7 View
Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?
02 March 2021 5,646 1 View
After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...
26 February 2021 5,947 3 View
As long as I know FAM is one of the brightest used fluorophore molecule when making a qPCR test. So why Am I getting this low RFU fluorescence mixed with kind of the normal signal I would get from...
22 February 2021 6,056 1 View
Hello, Here is my experiment: I transfect in a plasmid that has been cut once (a linear piece of dsDNA) into human cultured cells. I wait 48hrs to allow the cells to "repair" the plasmid making...
11 February 2021 6,225 3 View
I am very new to quantitative PCR and need some help for this technique. For my experiment, I need to perform a quantitative reverse transcription PCR. I extract the RNA from my pellet and...
06 February 2021 10,040 4 View
The parameters of the curve all seem acceptable, typically reactions results are above 0.9852, -3.1 to -3.6 slope, and 90-110% reaction efficiency. The results seem to be what I would expect from...
05 February 2021 1,316 3 View
Hi I am having issue's with Gibson assembly, seeing as I am new in the filed of cloning, expert feedback would be very much appreciated I am trying to clone two fragments (frag 1 [185bp, 145bp w/o...
30 January 2021 1,533 2 View
I want to detection 600 kDa of high molecular weight for 4% acrylamide gel. But i could not detect that i prepared 4% acrylamide gel... Could you tell me how to prepare a 4% acrylamide gel?
26 January 2021 1,469 3 View
Considering that we hypothetically designed an experiment, we have an x gene, and we have the mutant version of this gene with a single nucleotide mutation in one of the exons. What kind of an...
23 January 2021 9,765 12 View
Currently, I'm working on a DNA pol activity test, one of the variables is DNA pol concentration in the PCR/LAMP assay. For LAMP I found a clear protocol for the DNA pol in weights. However, for...
02 January 2021 9,992 3 View
Animals were fed DSS via drinking water and fecal samples were collected and DNA was isolated using Qiagen stool DNA isolation kit. The V4 region of extracted DNA was amplified with specific...
21 December 2020 1,879 3 View
Hey Everyone, This is my first post here. I am a first year graduate student. I am working on generating a standard curve using the cDNA I have generated this semester. It looks like I am getting...
15 December 2020 8,870 8 View
I intend to fuse my gene of interest (apoptin) with GFP using pCAMBIA1302 vector. I figured that I can do that by adding NcoI and SpeI restriction sites to 5' and 3' end of my GOI using PCR and...
14 December 2020 4,485 3 View
I am trying to predict age from DNA methylation. I am using conventional PCR master mix, but I didn't get my PCR product. Is it due to Master Mix? Should I use HotStar Taq Master Mix ?
09 December 2020 8,940 4 View
I have this problem. Today I used a new positive control of an isolated NC-1 strain in cell culture and the problem persists. I performed a serial dilution test to test the sensitivity of the...
09 December 2020 6,422 2 View
I did a PCR and inserted restriction sites on either side of my gene of interest. I then digested the PCR product and performed ligation into two different plasmids. 5:1, insert vector. I...
30 November 2020 1,631 16 View
Hello everyone, I hope you are very well. I am performing site-directed mutations with the QuikChange II kit. My first ones have a length of 50bp and a Tm of between 75 and 81 approximately. I...
10 November 2020 384 2 View
Hey everyone. I collected some leaf tissue samples from the plant Phragmites australis from which I am hoping to extra DNA for sequencing. I will be extracting DNA using Qiagen DNeasy Plant Mini...
09 November 2020 5,133 12 View