The PCR Master Mix is designed
for routine endpoint PCR for DNA amplicons in the range of 0.2–2kb
. PCR Master Mix allows you to set up your reactions in less than a minute—just add template and primers. Optimized conditions enable amplification of as few as 2 copies of target template.
What are some advantages of using a master mix?
- Save time: Blends are stored at +2 to +8°C and can be immediately used, no freezing or thawing is necessary.
- Improve results by reducing pipetting errors: Master mixes contain all the reagents required to perform PCR.
Why is it important to prepare a master mix?
The master mix usually includes DNA polymerase, dNTPs, MgCl
2
and buffer. Using a master mix
reduces pipetting and risk of contamination
, is convenient, saves time and preempts possible errors in mixing, making it ideal for high-throughput applications.
What is a PCR master mix and what is the advantage of using it?
Using a PCR master mix for PCR assays
provides faster setup with less pipetting
—the mix can be prepared once and divided among pipettes to save time. By reducing the scope for pipetting, there is less room for experimental error, a reduction in contamination, and less variability among tubes.
What is the importance of creating a master mix quizlet?
**The master mix contains a thermostable DNA polymerase, gene-specific forward and backwards primers, each of the 4 dNTPs, reaction buffer and MgCl2. **The mix will
drive the PCR, increase efficiency, and decrease the risk of contamination
.
What are the 4 major components of PCR master mixes?
The master mix usually includes
DNA polymerase, dNTPs, MgCl
2
and buffer
. Using a master mix reduces pipetting and risk of contamination, is convenient, saves time and preempts possible errors in mixing, making it ideal for high-throughput applications.
How do you make a master mix for PCR?
For a 25μl reaction volume:
PCR Master Mix, 2X 12.5μl 1X upstream primer, 10μM 0.25–2.5μl 0.1–1.0μM downstream primer, 10μM 0.25–2.5μl 0.1–1.0μM DNA template 1–5μl < 250ng Nuclease-Free Water to 25μl N.A.
Why is buffer needed in master mix?
Reaction buffer in the Mix
enhances specificity and efficiency of PCR
; it contains a buffer, which after addition of proper amount of MgCl
2
is optimal for majority of PCRs.
What does the master mix contain?
A master mix usually contains
a thermostable DNA polymerase, dNTPs, MgCl
2
, and proprietary additives in a buffer optimized for PCR
. Only template, primers, probes (if being used), and water, to make up the volume, need to be added.
What are the 3 types of PCR?
- Real-time PCR.
- Quantitative real time PCR (Q-RT PCR)
- Reverse Transcriptase PCR (RT-PCR)
- Multiplex PCR.
- Nested PCR.
- Long-range PCR.
- Single-cell PCR.
- Fast-cycling PCR.
What is quantitative real time PCR used for?
Quantitative PCR (Q-PCR) was used
to measure the amount of PCR product
. It is the preferred method to measure quantitatively the levels of transgenic DNA. Q-PCR is often used to determine the number of copies in the sample.
Why do PCR products differ in size?
Primers are never 100% specific
so they can always bind to a non-desired region of your template DNA, which would lead to a product with a size that’s different from the one you are expecting.
What do buffers do in PCR?
PCR is carried out in a buffer that
provides a suitable chemical environment for activity of DNA polymerase
. The buffer pH is usually between 8.0 and 9.5 and is often stabilized by Tris-HCl. For Taq DNA polymerase, a common component in the buffer is potassium ion (K
+
) from KCl, which promotes primer annealing.
What are the main purposes for adding a loading dye to the samples?
Loading dyes
impart color to the samples
, which visually facilitates the loading process. Last, the loading dyes increase the density of the sample, which ensures even loading in the sample well.
What are the components reagents required to complete a PCR reaction and what is their purpose?
Standard PCR reagents include a
set of appropriate primers for the desired target gene or DNA segment to be amplified
, DNA polymerase, a buffer for the specific DNA polymerase, deoxynucleotides (dNTPs), DNA template, and sterile water.
