PCR notes

To make stronger signal, I will mix all the primer targets together (IL10, Hdac3, Ccl3, beta-actin)

Here is signal

Here is negative control

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Negative control PCR stock (10 rxns)

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PCR stock (10 rxns)

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Deepak kit

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Primers (Don't know corresponding code, yet)

1. IL10
2. Hdac5
3. Ccl3
4. beta-actin

Primer ids

1. 0208661104
2. 0374221766
3. 0347861862
4. 0367609480

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CDC recommendation: https://www.fda.gov/media/134922/download

https://www.cdc.gov/coronavirus/2019-ncov/lab/virus-requests.html

https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html

https://www.idtdna.com/pages/landing/coronavirus-research-reagents/cdc-assays

https://www.quantabio.com/campaigns/letsfighttogether_ga?gclid=CjwKCAjw34n5BRA9EiwA2u9k3_H7ItLPpVG8GCRj42IUl9XJ3_7RCtN-ftmuuMUw3BMcGyAgsNKtPhoC_jIQAvD_BwE

Speeding it up

How to speed up the polymerase chain reaction https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496742/

Despite these improvements, what has not changed a great deal are the protocols used to run mainstream qPCR reactions. A quick perusal of the peer-reviewed literature reveals that current standard methods, if they are published at all, include near universal denaturation temperatures and times of 95 °C and 15–30 s, respectively, annealing/polymerisation times of 15 s–1 min and optional separate polymerisation times of 45 s–2 min. These add up to around an hour for a typical 40-cycle reaction. In addition to these times, the speed of a PCR run is also dependent on ramp rates, i.e. how fast the PCR instrument can change from one temperature to another and especially how fast the cooling process works. Hence any attempt to reduce significantly PCR run times must look at reducing both cycle times and minimising the differences in temperatures for the various steps.

More recently, super-fast PCR reaction times were achieved by increasing primer and polymerase concentrations to around 20-fold above typical concentrations, increasing annealing/extension temperatures to around 75 °C and reducing denaturation temperatures to below 90 °C, so allowing amplification of short PCR amplicons less than 15 s

• PCR amplicon sizes of around 100 bp are optimal, although the use of longer amplicons, such as UBC at 177, is possible.

• Since substitution of TTP with dUTP lowers nucleotide incorporation rate, even if its concentration is increased, master mixers that use dUTP should not be used if speed is the main consideration.

• If possible, amplicons with higher GC contents (around 60%) should be chosen since these templates have the highest extension rates.

• Two-step PCR is faster than three-step PCR. Extension rates are maximal at 5 °C below the Tm of the primers. Given that the optimal temperature of extension is between 70 °C and 75 °C, the fastest two-step PCR would occur with primer Tms at 65 °C or above.

If we have 3 different heat blocks it can be much faster

http://www.nextgenpcr.com/wp-content/uploads/2016/05/MBS-application-note-2-minute-pcr.pdf

Extreme PCR: Efficient and Specific DNA Amplification in 15-60 Seconds

https://pubmed.ncbi.nlm.nih.gov/25320377/

Background: PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown.

Methods: We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4-2.0 s at annealing/extension temperatures of 62 °C-76 °C and denaturation temperatures of 85 °C-92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring.

Results: Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15-60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%-95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles.

Conclusions: The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4-2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

Plastic Versus Glass Capillaries for Rapid-Cycle PCR

https://pubmed.ncbi.nlm.nih.gov/18476813/

Rapid-cycle PCR uses fast temperature transitions and minimal denaturation and annealing times of “0” s to complete 30 cycles in 10 to 30 min. The most popular platform amplifies samples in glass capillaries arranged around a carousel with circulating air for temperature control. Recently, plastic capillary replacements for glass capillaries became available. We compared the performance of plastic and glass capillaries for rapid-cycle PCR. Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area-to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6 degrees -7 degrees C. Rapid-cycle PCR was performed on two human genomic targets (APOE and ACVRL1) and one plasmid (pBR322) to amplify fragments of 225-300 bp in length with melting temperatures of 90.3 degrees -93.1 degrees C. Real-time amplification data, end-point melting curves, and end-point gel analysis revealed strong, specific amplification of samples in glass and complete amplification failure in plastic. Only the APOE target was successfully amplified by extending the denaturation and annealing times to 5 or 10 s. A 20 s holding period was necessary to reach target temperatures in plastic capillaries.

Under-three Minute PCR: Probing the Limits of Fast Amplification

https://pubmed.ncbi.nlm.nih.gov/21796289/

Nucleic acid amplification is enormously useful to the biotechnology and clinical diagnostic communities; however, to date point-of-use PCR has been hindered by thermal cycling architectures and protocols that do not allow for near-instantaneous results. In this work we demonstrate PCR amplification of synthetic SARS respiratory pathogenic targets and bacterial genomic DNA in less than three minutes in a hardware configuration utilizing convenient sample loading and disposal. Instead of sample miniaturization techniques, near-instantaneous heating and cooling of 5 μL reaction volumes is enabled by convective heat transfer of a thermal fluid through porous media combined with an integrated electrical heater. This method of rapid heat transfer has enabled 30 cycles of PCR amplification to be completed in as little as two minutes and eighteen seconds. Surprisingly, multiple enzymes have been shown to work at these breakthrough speeds on our system. A tool for measuring enzyme kinetics now exists and can allow polymerase optimization through directed evolution studies. Pairing this instrument technology with modified polymerases should result in a new paradigm for high-throughput, ultra-fast PCR and will hopefully improve our ability to quickly respond to the next viral pandemic.

Ultra Fast Miniaturized Real-Time PCR: 40 Cycles in Less Than Six Minutes

https://pubmed.ncbi.nlm.nih.gov/16807313/

We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the system's thermal constant of 0.27 s, we have achieved a heating rate of 175 degrees C s(-1) and a cooling rate of -125 degrees C s(-1). A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput.

Fast real time PCR

https://www.thermofisher.com/order/catalog/product/4351107?SID=srch-srp-4351107#/4351107?SID=srch-srp-4351107

1- vs. 2-Step RT-PCR You can perform reverse transcription (RT) and PCR in a single reaction (1-step) or in separate reactions (2-step). The reagent configuration you use depends on whether you are performing 1- or 2-step RT-PCR:

• In 1-step RT-PCR, RT and PCR take place in one buffer system, which provides the convenience of a single-tube preparation for RT and PCR amplification. However, you cannot use Fast PCR master mix or the carryover prevention enzyme, AmpErase® UNG (uracil-N-glycosylase), to perform 1-step RT-PCR.

• 2-step RT-PCR is performed in two separate reactions: First, total RNA is reverse transcribed into cDNA, then the cDNA is amplified by PCR. This method is useful for detecting multiple transcripts from a single cDNA template or for storing cDNA aliquots for later use. The AmpErase® UNG enzyme can be used to prevent carryover contamination.

Clean Up Your Act! How To Clean Up PCR Contamination

https://bitesizebio.com/20773/clean-up-your-act-how-to-clean-up-pcr-contamination/

The biggest source of PCR contamination is aerosolized PCR products, which are created when you open a tube or pipette amplified PCR product. Once these droplets are created, being small, they travel well. These tiny droplets easily spread all over your bench and equipment, where they can find their way into your next PCR and be amplified

Use a 10% bleach solution or DNA-away to wipe down your.

Bench top

Pipettes

Centrifuge

Vortex

Racks

Thermocyler lid and buttons