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Questions related to PCR Analysis
Hi all. We performed HRM analysis on a LightCycler 480, but our Roche software doesn't have the Gene Scanning module enabled. We are not capable of purchasing a $5000 software right now and would...
12 December 2014 1,510 1 View
I sequenced with both the forward and reverse primers in separate reactions (one tube forward primer + amplified DNA, and the other tube reverse primer + amplified DNA). The reverse reaction shows...
12 December 2014 2,953 1 View
I am doing a RT-qPCR of mouse retinas. Each retina is very small. Can I use right and left retinas seperately, or would I need to use both a right and left retina in one mix to get enough sample?
21 November 2014 2,652 2 View
11 November 2014 9,224 3 View
We are looking for a cheap way to do PCR for routine genotyping in mice ? What is the cheapest company ? What is the cheapest complete kit ?
11 November 2014 4,606 0 View
When I do PCR, I stock my primers in pure water, is it wrong?
11 November 2014 3,557 0 View
I am using the promega green master mix and the program I used: 1- 94C for 5 min 2- 45 cycle of 94C: 1:30min, anealing at 60.5C :30s , extetion at 72C : 2:30min 3- 72C for 10 min I tried to...
11 November 2014 2,194 21 View
I'm trying to optimize a polymerase chain reaction for detection of eubacterias, mycobacterias, among other things. I'm trying several different kits and I wanted to know the pros/contras of this...
11 November 2014 2,033 3 View
I'm using pACYC vector amd I'm doing double digestion with BamHI and HindIII for both of my vector and insert. 1) In my primer design, I only added one base pair before the restriction site...
11 November 2014 5,523 5 View
I did a PCR experiment and sequenced the product. The result is very surprising. I had it sequenced in both directions with forward and reverse primer respectively and aligned them together...
11 November 2014 4,438 11 View
I ask this question because I have found that the PCR efficiencies for my housekeeping and target genes are nearly identical when the former reaction uses 1:1000 diluted cDNA and the latter...
11 November 2014 1,463 11 View
I know a value of 1.8 is the purest form of DNA.
11 November 2014 3,093 7 View
Good Afternoon Everyone I am planning on purifying my PCR product and running three separate restriction digests on that product. The enzymes are BstUI (CG/CG), TaqI(T/CGA), HpyCH4IV(A/CGT). I...
10 October 2014 2,179 5 View
Are there any other protocols?
10 October 2014 1,249 8 View
Dear all, I am getting smeared bands on my PCR product of brachy podium DNA. I try hard to test DNA quality but in vain. NANO drop is showing good values for DNA, 260/280 ratio but very smeared...
10 October 2014 6,807 5 View
We have currently trying to add a GFP tag to a gene buy using homologous recombination with a PCR product. The marker we are using is G418 resistance and the transformations are done by a normal...
10 October 2014 3,810 5 View
I agree with most of the answers, but I´m facing a similar problem. I have a band (with the expected size) on negative control and I doubt that is contamination, because in the same set of...
10 October 2014 1,044 10 View
A denaturation cycle has a much higher temperature than the melting temperature for primers. Why do the primers never melt in the denaturation stage?
10 October 2014 8,389 8 View
10x Taq buffer and MgCl2 were provided separately, and I am using these at 1x concentration. Positive control and water controls are working good, but the target cDNA has not yet been amplified.
10 October 2014 5,672 11 View
Hello everyone, I am currently trying to find a reliable method for genotyping my mdx and wild-type mice. We have tried a couple of protocols already...
10 October 2014 678 1 View
I used from High Fidelity PCREnzyme Mix (thermo scientific:product number k0191) for PCR performing.My PCR product is 3131bp from HEK-293 cell line DNA.On the first day, I performed a PCR test but...
09 September 2014 5,197 14 View
Dear all, I have transformed my Ecoli with a clone of size (14.3kb). Plasmid alone is about 13.9kb and insert of size 432bp. In 1:3 ratio I got around 200 colonies, negative control plate (only...
09 September 2014 2,309 5 View
I started running nested pcr this week using Extract N Amp. This is my first time doing this, and after the second reaction, my water negative showed positive bands. I will look into contamination...
09 September 2014 1,453 4 View
I'm working on my reseach, the topics about gen barcoding of local sea cucumber in my city (i assumed that the sea cucumber is belong to Caudinidae).But i have a problem. Whenever I do my PCR...
09 September 2014 5,269 1 View