12
votes
Are there issues with filling PCR tubes to capacity?
(Also too long for a comment.) This question reminded me about an anecdote told by Arthur Kornberg concerning the approach to scaling up that was adopted by Reiji Okazaki (discoverer of Okazaki ...
12
votes
Are there issues with filling PCR tubes to capacity?
Too long for a comment: I would say it depends on the PCR. I have personally used maximum volumes of 50ul, if I needed to prepare higher volumes, I made one mix which subsequently got distributed to ...
11
votes
Good pipetting technique?
Here's some step-by-step advice on good pipetting technique:
Make sure the pipette is set for the correct volume
Ensure the tip is firmly attached
Keep the pipette vertical when pipetting
Slowly and ...
10
votes
What is the half-life of dNTPs at 95 °C?
Instead of searching for the stability of dNTPs, consider the stability of their constituent parts.
Caveat: ignoring the bonds between the different parts of dNTPs likely misrepresents the stability ...
9
votes
How does PCR mutagenesis add restriction site near the gene of interest?
This diagram would be easier for you to conceptualize if you instead used the actual nucleotides in the restriction sites and green GGAGAA region along with your gene of interest (GOI). I recommend ...
7
votes
Accepted
Basic question about multiplex PCR
I haven't done this exact experiment. I am just deducing from the known facts about PCR.
The product of PF and PR2 will serve ...
7
votes
Accepted
How are DNA segments selected in PCR?
Note: In your PCR program you always set extension time.
Case:
Product length = 500bp
PCR extension time = 50sec
Assuming that polymerase adds 1000 nt/min
Cycle 1:
Strand that binds FP: extends ~...
7
votes
Is Melanin a PCR Inhibitor?
Melanin is a potent inhibitor of PCR - when you use B16 cells (or any other cell line that produces melanin) you have to purify your sample from it. Unfortunately a simple Phenol-Chloroform extraction ...
7
votes
Is PCR a DNA cloning technique?
Short answer
All of your sources are correct as they are not mutually exclusive. PCR is used to isolate and amplify DNA to yield small quantities of pure target product. Gene cloning can subsequently ...
6
votes
Accepted
Equation for accurate prediction of PCR yield
An expected efficiency for a typical PCR is 80%, meaning each cycle multiplies the copy number of the targeted DNA sequence 1.58 times.
Firstly, it makes more sense to refer to the amount of DNA in a ...
6
votes
Accepted
Why do we need to amplify DNA sequences?
This is a question I also remember wondering about when I was younger in school. Now as a professional it's way too obvious to even explain. But i think it's an important and common question, which ...
5
votes
Real-time PCR result interpretation
You should try another housekeeping gene like GAPDH since the very definition of these genes is that they shouldn't change with your treatment
5
votes
Possible reasons for DNA getting stuck in well
There is one simple reason for that, your agarose gel is most likely too dense. Depending in the type of agarose, I would prepare a 0.5-0.6% gel at maximum.
Synbio gives this list for "standard" ...
5
votes
Accepted
DNA polymerase in PCR (polymerase chain reaction)
No, this does not happen, as the DNA polymerases used for PCR are DNA dependent. This means that they only synthesize DNA when it is bound to DNA. Even if your primers bind to the RNA, the polymerase ...
5
votes
Accepted
Walk me through microsatellite markers and PCR
It's actually very simple. You misunderstood the term polymorph in this situation.
A microsatellite is a sequence composed of a short repeated DNA motif. A polymorph satellite is then simply a ...
5
votes
Accepted
Does common PCR amplify genes regardless of what cells / barriers they are in?
There are many methods of DNA isolation, so it is difficult to make broad statements about all situations. Typically, though, during the DNA isolation protocol, essentially all proteins are denatured ...
5
votes
Accepted
Alternatives to PCR
Edited after clarifications in question,
Let's start with normal functions of both enzymes. Helicases separates DNA strands while polymerase synthesize DNA strands as shown in the following figure. (...
5
votes
Is Melanin a PCR Inhibitor?
I don't have much expertise in PCR (so please do not ask me technicalities). A research has proven that melanin is a PCR inhibitor as it forms complexes with thermostable DNA polymerase and inhibits ...
5
votes
Accepted
Why is only one primer used for DNA sequencing instead of two?
The reaction for the Sanger sequencing reacting contains beneath the normal dNTP (which are needed to make most of the DNA) the dideoxynucleotides, which lack the 3'-OH-Group and which are labelled ...
5
votes
Can primers for PCR be duplicated?
Short answer: You need to buy some more, but you need the sequence also for ordering.
Long answer: The Taq polymerase needs a piece of DNA (or RNA) to prime the reaction and be able to enlarge the ...
5
votes
Why PCR primers don't amplify beyond the target region?
How does DNA synthesis from the forward primer stop when it reaches the point where the reverse primer is?
Briefly, it doesn't. You get a lot of single-stranded long pieces of DNA (step 1 here, ...
5
votes
Accepted
Do the primers used for Covid-19 test work with all new strains?
I TRY to answer. First of all, no we cannot be 100% sure that the primers are effective against all strains.
However, the strains that are under media attention right now are due to variants in the ...
5
votes
Is it possible to use PCR to test for Machado-Joseph disease?
This paper describes an allele-specific PCR protocol to detect CAG repeat expansion in MJD patients:
Maciel P, Costa MDC, Ferro A, et al. Improvement in the Molecular Diagnosis of Machado-Joseph ...
4
votes
How are DNA segments selected in PCR?
I'm not completely clear when you say "what makes the replication terminate when the polymerase reaches the primer at the other end" since when you perform a PCR you go through three phases. The ...
4
votes
Accepted
Real time PCR normalization algorithm
First step is the calculation of efficiency, denoted by lets say $E_{gene}$. See this post for calculation of primer efficiency.
So the fold change for that gene will be calculated by $E_{gene}^{-\...
4
votes
Accepted
A question related to qPCR analysis
OK, let us start from the beginning. We know what makes one cell type different from another cell type is its expression - i.e. what genes are actually being transcribed into RNA. Therefore, there ...
4
votes
DNA barcoding and real-time PCR
You've done a pretty decent job of answering your own question, but there are a few things that can be elaborated on.
The general convention is to use the mitochondrial Cytochrome C Oxidase 1 (COI) ...
4
votes
Accepted
DNA extraction methods for hair?
I've been trying to figure out something that works for some time. I have recently tried 3 cheap protocols using commonly available lab reagents. I was using buccal samples (0.2 ml of spit).
1) ...
4
votes
Accepted
What happens if both forward and reverse primers have same Tm?
It is actually good if both the primers have the same Tm (Melting Temperature) because you would basically want both primers to anneal at your ...
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