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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 ...
rg255's user avatar
  • 16.1k
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 ...
acvill's user avatar
  • 8,286
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 ...
Ian_Schlander's user avatar
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 ...
Chris's user avatar
  • 51.6k
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 ...
AliceD's user avatar
  • 52.4k
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 ...
Patrick's user avatar
  • 116
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 ...
S Pr's user avatar
  • 6,202
6 votes
Accepted

What is the function of PCR in the whole genome sequencing?

PCR is used to replicate each isolated genome fragment, yielding several copies of each fragment in your DNA solution (that‘s called a library = a collection of fragments + adapters). This amplifies ...
markur's user avatar
  • 1,769
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 ...
another 'Homo sapien''s user avatar
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 ...
Chris's user avatar
  • 51.6k
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 ...
Chris's user avatar
  • 51.6k
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, ...
aaaaa says reinstate Monica's user avatar
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 ...
Fabio Marroni's user avatar
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 ...
acvill's user avatar
  • 8,286
5 votes

Primers spaning exon-exon junctions

See similar questions elsewhere. Looking into this paper on primer design, they write this: It is often necessary to choose the primers from the exon—exon junctions (E-E-jns), the regions of mRNA ...
Maximilian Press's user avatar
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 ...
WYSIWYG's user avatar
  • 35.5k
4 votes

PCR Mastermix Preparation

A PCR Master Mix is just a way to speed up your pipetting. Instead of preparing 10 different reactions of 20ul each (for example) you prepare one reaction of 200ul and then you split it into 10 tubes. ...
alec_djinn's user avatar
  • 3,098
4 votes
Accepted

Why PCR primers don't amplify beyond the target region?

They don't stop replication. It's just that you replicate your dna in a number of cycles, let's say 30. after which you'll have a approximatly 10^6 molecules of the amplified region and only a few ...
Jonas's user avatar
  • 533
4 votes

Do the primers used for Covid-19 test work with all new strains?

No, some strains contain mutations causing partial or complete failure of amplification of at least one of the test targets. Here's an FDA bulletin on the issue from January: https://www.fda.gov/...
Armand's user avatar
  • 1,711
4 votes

How to assemble three 60mer nts by pcr?

In order to assemble your TAG oligos by PCR you will need to redesign your primers so they are complementary to the TAG oligos as DNA polymerases work by adding nucleotides to the 3' end of a DNA ...
GaelC's user avatar
  • 466
4 votes

How are PCR tests for detecting COVID-19 performed on hamsters?

This is probably quite different from the example you're thinking of, but here's an example where they were using a hamster as a model organism for testing possible COVID-19 treatments for humans: ...
Bryan Krause's user avatar
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4 votes
Accepted

Do 0.2 vs 0.5 ml epitubes require different PCR machines, incubators, etc?

The answers are both yes and no. Many PCR machines come with the block designed to hold both 0.2 and 0.5 ml tubes (e.g. this one from Eppendorf**), or with swappable blocks (e.g. Bio-Rad C1000**), ...
bob1's user avatar
  • 11.6k
4 votes

Do 0.2 vs 0.5 ml epitubes require different PCR machines, incubators, etc?

Here's a top-down photo of the heating block from an Eppendorf 6331 Nexus Gradient MasterCycler (source): Most modern Eppendorf thermal cyclers have a similar block.
acvill's user avatar
  • 8,286
3 votes
Accepted

Why would you mix polymerases in PCR?

The Taq polymerase is a rather fast enzyme (the synthesis rate is around 1 kb/min), but the backdraw is that it has no 3'- 5'exonuclease activity which would enable to proofread the newly synthesized ...
Chris's user avatar
  • 51.6k
3 votes
Accepted

Why does Taq polymerase add 3' adenine overhangs?

Non-templated terminal addition by certain DNA polymerases is apparently dependent on the base stacking between the incoming base and the existing duplex (Fiala et al., 2007*). This was verified by ...
WYSIWYG's user avatar
  • 35.5k
3 votes

Good pipetting technique?

first of all i need to disclose i work for Andrew Alliance, but i answer for the sake of providing information and not for commercial purposes. What you experience depends - as we measured with our ...
user3172395's user avatar
3 votes

Purifying large amounts of PCR product

We had to do this frequently. What I would first recognize is that all of your current methods only work for at the small scale and that for you to purify mg quantities of DNA, you need to work with ...
bobthejoe's user avatar
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3 votes
Accepted

Choosing PCR conditions

You need to first build an initial protocol and then optimize it. The most important to determine the conditions is the polymerase used. Some polymerases work at higher or lower temperatures and will ...
Dart Feld's user avatar
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3 votes
Accepted

Manual Primer Design for a gene on the reverse strand

Not a stupid question at all. I was just as confused by this as a beginner in molecular biology. You get the hang of it after a while. :) I find it much more difficult to design primers from just one ...
CDB's user avatar
  • 1,836
3 votes

Can conventional PCR amplify DNA from different organisms from a specimen in a single step?

If you had a 100% BLAST hit for 3 species from a single amplicon sequence then it only means that region of DNA is conserved in the three species so you have at least one of those species present not ...
Artem's user avatar
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