Which DNA Polymerase should I use?
For standard PCR applications, we recommend using Thermoprime Taq DNA Polymerase which has all of the benefits of standard taq but with enhanced thermal stability. For long or high fidelity PCR, use Extensor Hi-Fidelity DNA PCR Enzyme, which has a synergistic mix of two enzymes enabling the amplification of DNA templates with double the yield and and four times the fidelity of standard Taq DNA Polymerase. To obtain the highest specificity we recommend chemically modified Thermo-Start DNA Polymerase to eliminate non-specific priming.
Is Thermo-Start Taq DNA Polymerase chemically or antibody modified?
The ThermoStart Taq DNA Polymerase is chemically modified to give high specificity and requires an activation step at +95°C for 15 minutes. The Thermo-Start Taq DNA Polymerase chemically modified enzyme is not activated until after the +95°C incubation making it more specific than antibody modified enzymes, which can be activated at around +60°C.
I am getting non-specific amplification. What product do you recommend for this?
Thermo-Start Taq DNA Polymerase is ideal for preventing non-specific priming. For greater stringency, the enzyme can also be activated in increments by omitting the initial activation step and replacing it with extended denaturation times for the first few cycles.
I have GC rich and difficult templates, what do you suggest?
The Thermo-Start DNA Taq Polymerase has a High Performance Buffer option that is recommended for difficult and GC rich templates and also increases specificity and yield. For long PCR products the Extensor Hi-Fidelity PCR with Buffer 2 for difficult templates is recommended.
What length of fragments can be amplified with Extensor?
Extensor Hi-Fidelity PCR Enzyme can amplify sequences >20kb. Two buffers are supplied with this enzyme, Buffer 1 for products up to 12kb and Buffer 2 for product >12kb.
Do your enzymes leave a T/A hangover for cloning?
All of our polymerases have ‘terminal transferase-like’ activity and can be used for T/A cloning. For the Extensor Hi-Fidelity Enzyme mix most of the activity comes from licensed Taq DNA polymerase. This means that despite the 3'-5' exonuclease (proofreading) activity present in the mix, the majority of PCR products will be generated with an A-overhang, although the T-A cloning efficiency would be slightly lower compared to PCR amplification with Taq alone. PCR purification is recommended straight after PCR for T-A cloning to stop the residual activity of the proofreading enzyme, which may blunt end the amplicon.
Will ReddyMix interfere with downstream applications?
The ReddyMix dye is inert and will not interfere with standard downstream applications, including restriction digests and cloning. Standard clean up should be used to remove the dye before running on capillary sequences.
What are the benefits of using a Master Mix?
The MasterMix option allows significant time savings in preparation and gel loading, there are fewer reagent handling steps giving reduced chance of errors and ensured reproducible results. The MasterMix can also be stored at +4°C for up to a month eliminating freeze thawing of individual components.
Should I use 1.1x or 2x Master Mix?
The choice of Master Mix concentration depends on the concentration of template and primers to be added. The final concentration of buffer, enzyme and dNTP’s is identical for both the 1.1x and 2x mixes. For the 1.1x Master Mixes you use 45µL of mix in a 50µL reaction and then typically 1µL of each primer and 3µL of DNA. For the 2x Master Mixes you only use 25µL of mix in a 50µL reaction, allowing sufficient volume for the inclusion of more dilute template, primers and any additives you may use. Sterile water can be added to make the reaction up to 50µL.
Can I get my Master Mix pre-aliquoted and what are the advantages?
PCR MasterMix is available pre-aliquoted into any of our PCR tubes or plates, reducing the handling steps and time even further. This is ideal for high throughput laboratories allowing the preparation of hundred of plates in minutes by simply adding template and primers. For low throughput, Master Mixes can be pre-aliquoted into tubes for easy storage without freeze/thaw.
Why doesn’t CytosALL require a DNase treatment step?
When cells are lysed with CytosALL Reagent the nuclei remain intact and are subsequently removed by centrifugation. Further treatment to remove genomic DNA is not required.
How long does the extraction procedure take?
A 5 minute incubation on ice is followed by a brief (1 min) centrifugation step in a microcentrifuge. The lysate can be diluted and used immediately or aliquoted and stored for future use.
What downstream applications is the extracted RNA suitable for?
The cytoplasmic lysate can be used in 1- and 2-step RT-PCR, 1- and 2-step QRT-PCR, miRNA detection by QRT-PCR and RNA amplification reactions where limiting amounts of RNA are amplified using a T7 or T3 RNA polymerase system. For applications such as northern blotting that require more concentrated samples the RNA from the lysate would need to be concentrated or purified.
What number of cells is this procedure optimized for?
The absolute number of cells is not as important as the ratio of cells to reagent volume. The optimal ratio is 600µL/106 cells. Fewer or more cells can be lysed with the corresponding change in reagent volume. For example, 60µL of the reagent should be used with 105 cells.
Can I scale the amount of CytosALL used depending on how many cells I add?
Yes, the reagent can be scaled up or down. It is recommended that the suggested ratio of cells to reagent be maintained to prevent any inhibition of enzymatic reactions and allow for linear amplification in QPCR.
What yield of RNA can be expected?
The yield of RNA will vary with the cell type and cell physiology. For example, with HeLa cells, depending upon the physiological status of the cell, 6-12µg of RNA /106 cells (as measured with a Ribogreen assay) can be expected.
How long are the lysates stable for at -20°C?
The lysates can be stored for at least 2 weeks at -20°C. Gel electrophoresis of RNA purified from stored lysates demonstrate no detectable degradation. We have successfully used lysates stored at -20°C for one month.
Is it possible to quantitate levels of RNA in these lysates by spectrophotometer?
Direct methods such as UV absorbance are not recommended as other cellular material in the lysate can absorb in this range. An approximation of the amount of RNA present can be obtained using a Ribogreen assay.
What are the benefits of the ABsolute Blue QPCR Mixes?
ABsolute Blue is a range of high quality QPCR Master Mixes which generate higher end point values and lower Ct values than the competition. ABsolute Blue contains an inert blue dye for easy visualization of the reaction. This is particularly important for using white plates that give superior QPCR results but lead to aliquoting and visualization problems with traditional colourless reagents.
What is the blue dye? Will it inhibit the QPCR reaction?
The dye is an inert blue dye that is proprietary to ABgene. No, it will not affect the QPCR reaction.
How do I compare ABsolute Blue QPCR mixes to competitor’s mixes?
Different competitor’s mixes should be tested on different plates and an optimized protocol for each mix should be used. For ABsolute Blue it is critical that the Thermo-Start enzyme activation of +95°C for 15 minutes is carried out.
Do I need to DNase I treat my RNA before running QRT-PCR with Verso?
No, the Verso kits include RT Enhancer that, when included in the cDNA synthesis reaction, removes contaminating DNA eliminating the need for DNase I treatment.
Should I do 1-step or 2-step QRT-PCR?
1-step QRT-PCR is a quick and simple reaction with only a few pipetting steps which reduces the chance of pipetting errors and contamination and allows minimal hands on time for your results. The 2-step QRT-PCR requires more time and plastics consumables but cDNA can be stored for future QPCR reactions, the RT and QPCR reactions can be separately optimized and there are multiple priming options available.
Which RNA primers should I use for QRT-PCR?
For 2-step kits, random hexamers generate the most diverse pool of cDNA, whereas oligo-dT primers anneal only to mRNA poly-A tails and thus will be biased towards the 5’ end. A 3:1 blend of random hexamers to anchored oligo-dT is recommended to take advantage of the benefits of both primers. For 1-step kits, gene-specific primers are required and should be designed to span introns to stop genomic contamination. Shorter primers than standard PCR should also be used, ideally between 60-300bp.
Are ABgene plastic consumables sterilised?
All ABgene plastic consumables are produced in a state of the art ISO Class 8 (100,000) cleanroom facility. The injection moulding process involves heating the plastic to 220ºC, effectively destroying potentially contaminating DNA and endotoxins. All plastic consumables are certified DNase, RNase, human DNA and endotoxin free. As such, they do not require sterilisation.
Are ABgene plastic consumables DMSO resistant?
ABgene plastics are made from polypropylene, which is resistant to DMSO at room temperature and below (but not at elevated temperatures).
Why are opaque white plates recommended for QPCR?
White plates enhance geometrical optics (taking full advantage of the reflective/refractive properties of light). All light is consistently reflected off the plate surface rather than passing through the polymer and causing inconsistent refraction patterns. As well, white plates eliminate cross-talk between wells. For these reasons, white plates consistently provide QPCR results with the lowest well-to-well variance and highest sensitivity. It should be noted, however, that natural plates are successfully used and will give good results.
Why are frosted plates not recommended for QPCR?
Frosted plates have an uneven surface, causing uneven refraction of light and therefore high well-to-well variance. As consistency is a prerequisite to accuracy, variance should be kept to a minimum. Therefore frosted consumables are not recommended for QPCR.
Do you have a PCR plate suitable for use on Fast machines?
Yes, the AB-1900 has been specially designed with a low profile and thin walls to give optimum efficiency on the Fast Block machines.
Is the 384-well Diamond PCR Plate suitable for PCR applications? Do I need to reoptimise my reaction for these plates?
The Diamond 384-well PCR Plates are constructed from a extremely rigid, crystal clear polymer making it ideal for high throughput PCR applications and robotic plate handling. This polymer is optimised for use with PCR cycling conditions including the use of the heated lid at standard PCR temperatures and as the Diamond plates have the same heat transfer rate as standard polypropylene plates no further optimisation is required.
Do I need to change my PCR protocol when switching from 96 well plates to 384 well plates?
Yes, the 384-well plates are much lower profile than the 96-well plates, which decreases the distance between the heated lid and the samples. Having the heated lid set at the standard temperature of +110ºC can heat up the samples and skew the reaction, to stop this effect the lid temperature should be lowered between +90 and +100ºC. The annealing temperature may also need to be lowered by a couple of degrees, so running a temperature gradient is recommended.
I use very small volumes in my PCR. How can I make cycling more efficient?
ABgene produces a range of low profile plastic consumables (Low Profile Thermo-Fast 96 AB-0700; Low Profile 8-tube Thermo-Strips, AB-0771; Low Profile 12-tube Thermo-Strip, AB-0847) which are ideal in this situation. The shortened tube height with the low profile design reduces the dead space between the sample and heated lid of the thermal cycler. This minimises sample condensation during PCR and therefore increases reaction efficiency.
My plates often buckle out of shape and I find them hard to get off the PCR machine. Do you have any products that counter this?
The SuperPlate range of plates combines an innovative frame design with a specially selected polymer that produces a plate that virtually eliminates the warping and robotic handling problems seen with traditional PCR plates. These rigid plates retain all of the advantages of polypropylene plates allowing for optimal heat transfer and maximum sample recovery.
Do you have PCR plates suitable for use with robotic systems?
ABgene’s SuperPlate Skirted PCR Plate (AB-2800) and Thermo-Fast 384 (TF-0384) for high throughput are ideal for this application. The skirted design of these plates aids compatibility with automated systems and provides extra space for labelling or bar coding. Eight strategically placed holes in the skirt also aid plate positioning and removal from the thermal cycler block.
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