PCR Preparation

INTRODUCTION

The Polymerase Chain Reaction (PCR), one of the most powerful laboratory techniques ever discovered, is an in vitro technique in which a particular DNA sequence is amplified. The technique is based on the mechanism of DNA replication in vivo in whichdouble-stranded DNA is first denatured to single-stranded DNA, then duplicated, and the process repeated to yield multiple copies. PCR is extremely sensitive, and highly specific and due to the exponential nature of the reaction, able to yield high amounts of target DNA or accurately detect very small amounts of a specific target.

A general PCR workflow includes the following 3 steps:

  • 1) Choosing Target Substrates: The choice of the target DNA is dictated by the specific experiment.  Primers are then designed based on the DNA sequence and tested in silico for binding specificity.
  • 2) Setting Up the Assay:Once you have chosen the appropriate substrate and PCR primer sequences, the basic components of the reaction include: Polymerase, MgCl2,
 dNTPs, 
Forward/Reverse Primers, Target DNA, 10x Reaction Buffer and distilled water.
  • 3) Selecting Assay Conditions:PCR amplification conditions are composed of the total number of cycles to be run and the temperature and duration of each step in those cycles. The decisions are based on the amount of DNA target material you start with and how many copies of the PCR product are required.
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PCR workflows can vary dramatically depending on application, and unless you are using a DNA polymerase designed for Hot-Start PCR, the work MUST be performed at 4°C or less to reduce nuclease activity and prevent degradation. NOTE: Even when using Hot-Start PCR, if low yield, no amplification or non-specific amplification is observed then this can often be eliminated by adopting a pseudo-hot start protocol, i.e Hot-Start set-up conducted at 4°C. BioCision's products can be incorporated into a standard PCR workflow at two key stages:  nucleic acid isolation/sample preparation (particularly for q-PCR and RT-PCR in which mRNA is isolated), and the PCR assay set-up.
 

NUCLEIC ACID SAMPLE ISOLATION

The first step in many PCR workflows is the isolation of DNA or RNA from other cellular components.

Current Method - Tubes in Ice

15 sample tubes inserted directly into ice, a common method of keeping samples cool during the nucleic acid extraction setup. This method, however, results in unorganized samples, variable sample temperature due to uneven, insulating air pockets in the crushed ice, and an increased risk of sample contamination due to microbes and nucleases present in ice.

CoolRack® Method

The same 15 tubes in a thermo-conductive CoolRack M15 tube module, which is made of an alloy that rapidly and uniformly adapts to <4℃. When resting on ice, the CoolRack module will keep all samples organized and at a uniform temperature.  Using a CoolRack module on ice permits optimal nucleic acid extraction, while ensuring reproducibility.

Ice-Free CoolBox™ XT Method

The same 15 microtubes shown above are in an ice-free CoolBox™ XT Microtube workstation. Samples are maintained at ~ 1°C for over 10 hours with an internal, reusable cooling core.  Ice and ice-related problems such as unorganized, wet samples; risk of contamination due to microbes and nucleases present in ice; and variable sample temperatures are eliminated, when the CoolBox XT workstation is used.

DNA: Many commercial kits for DNA suggest that room temperature is sufficient for DNA isolation. However, it is well known that reduced yields and poor quality may occur if samples are collected, stored and handled improperly. It is highly recommended that all samples prior to DNA isolation be kept at 4°C in a CoolRack module to ensure the temperature is uniform and to maintain integrity of the DNA. Where reagents such as RNase A are required to be stored on ice, we recommend that a CoolRack module be used in order to ensure uniform temperature to all tubes.  Moreover, it will help reagents remain organized and there is a reduced risk of nuclease contamination.

RNA: PCR is commonly used to quantitate the amount of a particular gene transcript expressed and the starting material is RNA (q-PCR). In this case, a preliminary reverse transcription reaction is required to convert the mRNA into DNA. It is crucial to note that mRNA is significantly less thermostable than DNA, and that when working with mRNA, extreme care should be taken in handling the reactions. In order to accurately analyze these transcripts, it is important to extract high-quality RNA from the cells or tissues of interest with minimal degradation.Keeping samples cold (4°C) prior to RNA extraction IS REQUIRED as it inhibits RNase activity to safely preserve RNA. Click here to see the detailed BioCision RNA isolation protocol which describes how the CoolBox™ XT workstation and CoolRack® module can be incorporated into a general RNA extraction workflow.

 

PCR ASSAY PREPARATION

It is important that all PCR reagents be kept at 4°C during the assay set-up. PCR primers are especially sensitive to temperature and may degrade due to the 3’ to 5’ exonuclease activity of Pfu DNA Polymerase or PCR Enzyme Mixes. Additionally, dNTPs can be sensitive to multiple freeze/thaw cycles.

Snap freezing in dry iceCurrent Method - Directly in Ice

This image shows a PCR plate and reagent tubes, inserted directly into ice, a common method of keeping PCR samples cool during assay set up. This method, however, results in variable sample temperature due to uneven, insulating air pockets in the crushed ice, and an increased risk of sample contamination due to nucleases and/or microbes present in ice.

Snap freezing in dry ice using a CoolRackCoolRack® Method

The PCR plate and microtubes shown above are seated in a CoolRack PCR96 plate module and CoolRack M6 microtube module. These modules are made of a thermo-conductive alloy, which quickly adapts to the ice temperature (<4°C in 60-90 seconds) and keeps all tubes and plates uniform (+/- 0. 1°C), stable, organized and dry. Many PCR protocols are significantly enhanced with the addition of the CoolRack PCR and microtube modules to the workflow.

Snap freezing in dry ice using a CoolRack

Ice-free Coolbox™ XTMethod

The PCR plate and microtubes are kept cold in an ice-free CoolBox™ XT cooling workstation. Samples are maintained close to 1°C for over 10 hours with an internal, reusable cooling core. Ice and ice-related problems such as unorganized, wet samples, risk of contamination due to microbes and nucleases present in ice, and variable sample temperatures are eliminated when the CoolBox XT workstation is incorporated into the PCR workflow.

NOTE: Temperature consistency from well-to-well becomes even more critical in any type of quantitative experiment where the amount of RNA in one well is compared with the amount of RNA in another well. Any temperature differences across the reaction plate during experimental set-up can introduce variations that can affect the underlying quantitative differences in the samples that are being measured.

 

ELIMINATING ICE FROM BENCH TOP SAMPLE COOLING

CoolBox XT ice-free cooling workstations provide an unrivaled alternative to ice-based cooling.  CoolBox XT does not require electricity or batteries, but through the use of an internal cooling cartridge - the XT Cooling Core - samples can be kept uniformly and reproducibly cool (0.5 to 4℃) for a minimum of 10 hours and up to 16 hours.  And, the compact design takes up half the space of a conventional rectangular ice pan, saving precious bench top space.

CoolBox XT is easy to assemble and easy to use.  Simply store the XT Cooling Core in a -20℃ freezer and insert it into the CoolBox XT base when ready to work.  Place a room temperature (or pre-chilled, if preferred) CoolRack tube module or a CoolSink plate module on the XT Cooling Core and allow sample module to equilibrate to < 4℃.  Then insert your tube or plate samples into the CoolBox XT sample module and keep them cold for hours.

 

• Keeps samples cold up to 16 hours with the lid on, 10 hours with lid off

• Magnetized parts for easy assembly and secure handling

• XT Cooling Core features an embedded LCD temperature indicator for visual temperature assurance

• Easy to clean and sterilize with bleach solution or alcohol.

• Ideal for use in a cell culture hood or other area where ice is prohibited or a contamination concern

CoolBox XT Performance:


 

(Left) Microfuge tubes in a CoolBox XT ice-free cooling workstation.  Samples stay uniformly ~1℃ for up to 10 hours with the lid off (center), and for over 16 hours with the lid on (right).