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Can I use PCR buffer instead of cDNA synthesis buffer?

Can I use PCR buffer instead of cDNA synthesis buffer?


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I am using Fermentase First strand cDNA synthesis kit but its buffer is over. I need to work today but I have not access to any sorce currently. I have PCR buffer in the lab. Can I use PCR buffer instead of cDNA synthesis buffer? What are your other suggestions? Thank you


This page has the recipe for 10X M-MuLV RT buffer:

  • 500 mM Tris-HCl
  • 750 mM KCl
  • 30 mM MgCl2
  • 100 mM DTT
  • pH 8.3

If you have the basic reagents, I suggest making that.


Extracellular vesicle isolation frequently asked questions

The Capturem extracellular vesicle isolation kit provides a complete solution for the simple and rapid isolation of extracellular vesicles (EVs e.g., exosomes) from various biological fluids, such as plasma, serum, urine, milk, saliva, cell-conditioned media, and cerebrospinal fluid. We offer two sizes of the kit&mdashmini and maxi formats&mdashso you can purify EVs from a range of sample volumes.

For any questions not answered below, please see the user manuals and other documents accessible through the product table on this page. Please contact us if you can't find the answer to your question.

What binding compound is used in Capturem EV columns?

Capturem EV spin columns contain membranes that are functionalized with a lectin-based substrate that binds extracellular vesicles.

How is the Capturem EV method different from EV isolation using ultracentrifugation?

  • Ultracentrifugation separates particles according to density. Therefore, it isolates a mixed population of vesicles of varying sizes, along with non-EV contaminants, such as proteins like albumin. Due to the high speed, this process is quite harsh and often damages EVs.
  • The Capturem extracellular vesicle isolation kit specifically isolates EVs that are less than 200 nm in diameter. The process is gentle, and EVs remain intact for downstream applications such as electron microscopy.

Why is total protein concentration lower with the Capturem extracellular vesicle isolation kit than with ultracentrifugation?

Capturem EV columns provide a high-purity EV sample that is free of contaminating proteins. Ultracentrifugation yields a sample that includes impurities, such as albumin. Therefore, ultracentrifugation will give a higher total protein concentration due to these impurities.

Can I use the purified sample directly for my downstream applications?

Our kit elutes EVs using a phosphate-based buffer containing organic salts. Eluted particles can be directly used for physical particle analysis, such as nanoparticle tracking analysis (NTA) and labeling for cellular uptake assays. If your downstream application involves protein and/or RNA extraction, it will be necessary to sonicate or desalt the eluted samples first.

Can I use a different elution buffer?

Yes, you can use competitive elution using a mannose or glucose buffer. However, this will lower your EV yield, and you will need to determine if these buffers are compatible with your downstream applications.

Extracellular vesicle yield with different elution buffers. Extracellular vesicles were isolated from the same amount of plasma (200 µl) using Capturem extracellular vesicle isolation columns (mini). Contents were eluted with 100 µl of 1) 200 mM methyl &alpha-D-mannopyranoside in 150 mM NaCl, 2) 1 M glucose in 150 mM NaCl, or 3) Capturem EV elution buffer. Panel A. NTA was used to evaluate the concentration and size distribution of EVs obtained with the different elution conditions. Panel B. Overall yield (total number of EVs) from 200 µl plasma is highest using the Elution Buffer provided with the kit.

Can I sonicate the purified EVs for western blot analysis?

Yes. We sonicate our samples at 20°C for 5 min in a water sonicator (Ultrasonic Cleaner, JSP). If you are using a different sonicator, it might require optimization.

How can I desalt the purified EV samples?

We recommend the following steps:

  1. Load the entire sample obtained with the mini kit (or 500 µl of the sample obtained with the maxi kit) on an Amicon Ultra-0.5 Centrifugal Filter Unit (either 3-kDa or 10-kDa MWCO, supplied by the user). If the sample volume is less than 500 µl, add water to the filter unit to bring the volume up to 500 µl. Centrifuge at 14,000g for 5&ndash30 min and discard the flowthrough.
  2. Add water to the filter unit to bring the volume up to 500 µl, and centrifuge at 14,000g for 5&ndash30 min.
  3. Transfer the retentate (sample in the filter) to a new 2-ml collection tube (supplied by the user).
  4. For the sample obtained with the maxi kit, repeat Steps 1&ndash3 with the remaining 500 µl of the sample.

How much protein and RNA can I expect to purify from the EV samples?

This number can vary depending on the input sample type, volume, and conditions. In our experience, for EVs recovered from 1 ml of plasma, yielded approximately 1 ng of RNA and 5 µg of protein from the EV cargo.

How does the EV yield from the maxi column compare to the yield from the mini column?

From 1 ml of plasma, the Capturem EV maxi column gives an EV yield that is 10 times greater than the mini column. At its maximum capacity, the Capturem EV maxi column gives an EV yield (total number of EVs) of

What is the purpose of the pre-clearing column?

The pre-clearing column removes any large membrane fragments, apoptotic bodies, smaller cell fragments, etc., which would otherwise clog the Capturem column and prevent EV isolation.

What is the purpose of the Amicon filter unit, and why is it required?

The filter unit removes nonspecific protein aggregates, lipoproteins, cytokines, etc., resulting in EVs of higher purity as measured by fluorescent nanoparticle tracking analysis.

When using the mini kit, which Amicon filter for collecting sample supernatant do I need to purchase separately?

Purchase Amicon Ultra-0.5 Centrifugal Filter Units (Thermo Fisher Scientific, Cat. # UFC510024), which have a 100-kDa MWCO. Please make sure to use the filters as described in our protocol.

Can I treat Capturem-isolated EVs with Proteinase K to remove the bulk of proteins from samples instead of using an Amicon filter?

You can use Proteinase K, but it may result in partial degradation of proteins exposed on the surface of the EVs and therefore affect your ability to detect these proteins in your downstream analysis.

For the maxi columns, can I use a swinging-bucket or fixed-angle rotor?

Yes, you can use a swinging-bucket rotor or a fixed-angle rotor.


Reverse Transcription (cDNA Synthesis)

The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.

Many RTs are available from commercial suppliers. Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. M-MuLV Reverse Transcriptase lacks 3´ &rarr 5´ exonuclease activity. ProtoScript ® II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.

The use of engineered RTs improves the efficiency of full-length product formation, ensuring the copying of the 5' end of the mRNA transcript is complete, and enabling the propagation and characterization of a faithful DNA copy of an RNA sequence. The use of the more thermostable RTs, where reactions are performed at higher temperatures, can be very helpful when dealing with RNA that contains high amounts of secondary structure.

For help comparing the RTs and cDNA Synthesis reagents available, view our RT/cDNA Synthesis selection chart.

Choose Type:

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected]

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.


Application of cDNA:

cRNA contains a sequence of DNA only to code DNA. Using the reverse transcriptase quantification PCR, the amount of the particular transcript or mRNA or the gene of our interest can be estimated.

It is also used for gene cloning and transformation experiments.

The cDNA library is also used to study the expression of eukaryotic DNA by inserting it into a prokaryotic cell.

Heterologous expression:

The cDNA synthesis is applicable in the process of heterologous expression in which a protein is expressed in a type of cell that is not able to synthesize that protein naturally.

One of the important use of constructing cDNA is to clone low copy number genes.


Results & Discussion

A full length homeobox D13 1.1 Kb cDNA was generated by DF1 (HoxD13 forward primer1) and DR2 (HoxD13 reverse primer2) primers using genomic clone sequences as shown in fig 1. Restriction endonuclease digestion of this PCR product with Ppum 1 showed correct expected size fragments of 519, 316 and 265 bps respectively. The full length D13 clone in PCR ® II vector (Invitrogen) was digested with three restriction endonucleases that resulted in correct expected fragment size pattern as shown in fig 2. Furthermore, we sequenced this D13 clone using T7 primers to ascertain the correct open reading frame for this gene. No mutations were observed in D13 cDNA as a consequence of PCR manipulation.

Full length HOXD13 cDNA PCR product analysis. 1) 1.1 kb full length homeobox D13 cDNA generated by using DF1 and DR2 specific primers. 2 and 3) 100 bp ladder (Gibco BRL Life Technologies, Rockville, MD) 4). Restriction endonuclease digestion of PCR product with Ppum 1 showing correct expected size fragments of 519, 316 and 265 bps. respectively 5). 1 kb ladder (Gibco BRL Life Technologies, Rockville, MD) 6-8). are restriction digestion fragments of full length HOXD13 cloned in PCR II (vector) with Eco R1 (1.1 kb), Kpn 1 (381 bp) and Sma 1 and Bam H1 (826 bp) respectively.


Random primers are short oligonucleotides that have a random sequence. Since they are usually 6 nucleotides in length, they are often known as random hexamers. The random sequence enables the unbiased binding of primers to anywhere on most types of RNA including rRNA and small RNAs. Since they bind throughout, the majority of the gene products should be covered, therefore increasing the chance of amplification during PCR.

Oligo(dT) primers are composed of stretches of deoxythymidine. They are available in various lengths, with the most common being 12-18 nucleotides long. Oligo(dT) primers are designed so that they bind to the complementary poly(A) tails of messenger RNA (mRNA). Therefore, oligo(dT)s are only useful in cDNA reactions when mRNAs are the target for the downstream application. They will not anneal to non-polyA mRNA fragments, such as 18S rRNA. Also, bear in mind that if your PCR primers are designed near the 5’ end of a gene, the use of oligo(dT) primers for cDNA synthesis is not the best idea. Since these primers anneal to the 3’ end of mRNA, the 5’ end may not be present in the extended product, especially if you are investigating a large gene.


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Can I use PCR buffer instead of cDNA synthesis buffer? - Biology

In commonly used RNA isolation methods, the absence of genomic DNA is still a challenge. Particularly for downstream quantitative RT-PCR (qRT-PCR, co-amplified genomic DNA can lead to nonspecific results.

1.) Detection of genomic DNA in RNA samples

Different techniques have been used to monitor contamination of RNA by genomic DNA. Large amounts of contaminating DNA can be detected using OD measurement (producing a 260/280 nm ratio far below the optimum value of 2), or via agarose gel electrophoresis, where very high molecular weight nucleic acid bandings are obtained.

Nevertheless, in sensitive downstream applications like qRT-PCR, genomic DNA traces, not detectable by the above methods, can also produce nonspecific results. This is because both reverse transcribed mRNA (cDNA), as well as the contaminating genomic DNA, can serve as template for the subsequent PCR amplification. The result of this co-amplification is one or more additional PCR fragments that can be visualized via agarose gel electrophoresis based on their distinct molecular weight (see Figure 1).
One strategy to avoid this co-amplification is a PCR primer design that span exon boundaries.
This primer design strategy includes one or more intronic sequence motifs in the genomic DNA shifting the amplification efficiency of the PCR reaction toward the cDNA template. The specificity of the qRT-PCR assay should be monitored using a reverse transcriptase minus control reaction in the qRT-PCR setup. This control reaction is treated and handled similar to the other qRT-PCR reactions, with the exception that instead of the reverse trancriptase enzyme, water is added to the reaction mixture. PCR fragments amplified in this control reaction indicate contaminating genomic DNA.

Figure 1: Overview of first strand cDNA synthesis with different types of RT primers.

A GAPDH (lanes 1, 3 and 5) and RP49 (lanes 2, 4 and 6) gene specific PCR was performed including a control reaction lacking the reverse transcriptase enzyme (lanes 5 and 6). The PCR fragments in lanes 5 and 6 indicated by arrows, are of higher molecular weight based on the included intron sequence, and therefore indicative of contamination of the RNA template with genomic DNA.

2.) DNase I treatment
For removal of genomic DNA from RNA samples, a DNase I treatment is recommended. The DNAse I enzyme is classified as an endonuclease which is able to digest single and double-stranded DNA into single bases or oligonucleotides. The enzyme is DNA specific without negative effect on the integrity of the remaining RNA (Vanecko and Lasbowski, 1961).

DNase I treatment can be performed at the same time as the RNA isolation. This convenient procedure is used in the High Pure RNA Isolation Kit. The DNase I enzyme is included in the kit, and is directly applied during the spin column isolation procedure. The RNA is bound to the silica fleece in the presence of chaotropic salts, and the DNase I is directly applied and incubated with the purified RNA on the spin column. The digested DNA is subsequently washed from the spin column, leading to pure RNA eluted from the spin column.

In order to protect the isolated RNA, the DNase I Enzyme used should be of RNase-free quality. The recombinant DNase I Enzyme, RNase-Free from Roche is not only guaranteed to be RNase-free according to the strict current quality control, but is furthermore free of any animal components, such as contaminating DNA.

In cases of very high levels of endogenous genomic DNA, treatment with DNase I during the isolation process can still leave residual contaminating DNA in the isolated RNA. In this case DNase treatment can be performed after the RNA isolation.

Tip: As a rule of thumb for the DNase I digestion, use one unit of DNase I per 1 to 5 μg of total RNA in a 50 μl total volume incubated for 20 minutes at +25 to +37°C.

After the additional DNase digestion step an additional purification of the RNA from the DNase I enzyme is mandatory. This purification can be done by a cleanup procedure using the High Pure RNA Isolation Kit following the kit protocol (2.2 Isolation of Total RNA from Cultured Cells). Prior of the cleanup procedure the sample volume should be increased to 200 μl using the elution buffer included in the kit.
Note that in this case, the on-spin column DNase digestion step should be omitted from the protocol mentioned in the package insert.
The main benefit of this method is its ease of use, lack of toxic substances as well as the high recovery of total RNA.

Figure 2: Genomic DNA digestion by DNase I treatment.

A GAPDH (lanes 1, 3, and 5) and RP49 (lanes 2, 4, and 6) gene-specific PCR was performed including a control reaction lacking the reverse transcriptase enzyme (lanes 5 and 6). The PCR fragments in lanes 5 and 6 indicated by arrows, are of higher molecular weight based on the included intron sequence, and therefore indicative of contamination of the RNA template with genomic DNA.


A) Amplification of a GAPDH fragment by PCR.
B) Minus-RT control reaction with and without DNase I treatment. RNA was isolated from 1 x 106 K562 cells (human lymphocytes) using the High Pure RNA Isolation Kit with a subsequent amplification of a GAPDH-specific PCR fragments (lanes 1 to 6). No unexpected PCR fragment were obtained, indicating the absence of genomic DNA in lanes 1 to 4 (+DNase treatment), whereas those samples lacking the DNase treatment showed PCR products produced from contaminating genomic DNA (lanes 5 and 6) serving as template in the PCR amplification.
Tip: Even after DNase treatment, a subsequent PCR should be performed using exon-overlapping primer design. Also a reverse trancriptase minus reaction should be included as a control reaction for monitoring amplification specificity.

3.) Incomplete DNase digestion.
Very high levels of endogenous genomic DNA can still leave residual contaminating DNA in the isolated RNA. In case an additional DNase digestion step with subsequent cleanup is not an option, the amount of starting material should be reduced (i.e., 106 cultured cells, 500 μl whole blood, 108 yeast cells or 109 bacterial cells are maximum for the High Pure RNA Isolation Kit).
Another possible cause for incomplete DNA digestion is that the activity of the DNase I Enzyme can be decreased by suboptimal buffer conditions. The DNase I enzyme requires different divalent cations, both Mg 2+ as well as Ca 2+ , for optimal performance.
Tip: For optimal performance of the DNase I digestion the concentration of nucleic acid should be approximately 100 μg/ml.

Occasionally, the additional DNase I treatment, and specifically the heat inactivation of the DNase I enzyme, can negatively effect subsequent RT-PCR results. High temperatures necessary for the inactivation of the DNase I enzyme can also lead to RNA degradation. The heat inactivation of the DNase I enzyme requires an incubation at +75°C for 15 minutes. Since RNA molecules are heat sensitive (Huang et al., 1996), this incubation time should be as short as possible.

1. Chomczynski, P, Sacchi, N (1987). Anal Biochem. 162, 156-9.

2. Huang, Z, Fasco, MJ, Kaminsky LS (1996). BioTechniques 20, 1012-20.

3. Moore, S (1981). In: The Enzymes (Boyer PD, Ed.). Academic Press, New York.


Can I use PCR buffer instead of cDNA synthesis buffer? - Biology

‘Omic’ technologies, which adopt a holistic view of the molecules that make up an organism, are aimed primarily at the global detection of genes (genomics), mRNA (transcriptomics), proteins (proteomics) and metabolites (metabonomics) in a given biological sample. Application of genomic techniques in the field of diagnostic microbiology has limitations as genomics targets microbial organism-specific nucleic acids therefore, a positive result can occur with both active and inactive microorganisms. Transcriptomics has progressed along with advances in microarray and real-time reserve transcriptase PCR technology, while proteomics and metabonomics have benefited greatly from the increasing sophistication of mass spectrometrical techniques that detect protein and metabolic analytes. Together, transcriptomics, proteomics and metabonomics are helping to address questions about gene expression, thereby providing ‘functional’ diagnosis and assessment of microbial infections.

DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses

The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA from living bacteria.DNase I has been used mainly in the molecular biology field for the removal of bacterial genomic DNA contamination in samples for further RNA analyses (Wang et al., 2002).Suggesting that a sample containing free DNA, eDNA, and live and dead cells is exposed to DNase I, nucleic acids from living cells should be protected from the action of the enzyme due to its intact cell membrane.

Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems.

Identification of a third EspA-binding protein that forms part of the type III secretion system of Enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli utilizes a type III secretion system to deliver virulent effectors into cells. The secretion apparatus comprises a membrane basal body and an external needle complex of which EspA is the major component. An l0050-deletion (ΔL50) mutation was found to impair type III secretion and bacterial adherence. These phenotypes and the localization of the gene product to the inner membrane support the hypothesis that L0050, renamed EscL, forms part of the secretion apparatus. Furthermore, in ΔL50, the amount of EspA present within the cell lysate was found to have diminished, whereas the EspA co-cistron-expressed partner protein EspB remained unaffected. The decreased EspA level appeared to result from instability of the newly synthesized EspA protein in ΔL50 rather than a decrease in EspA mRNA. Using both biochemical co-purification and a bacterial two-hybrid interaction system, we were able to conclude that EscL is a third protein that, in addition to CesAB and CesA2, interacts with EspA and enhances the stability of intracellular EspA.


Affiliations

Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology, RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan

Kazuki Kurimoto, Yukihiro Yabuta, Yasuhide Ohinata & Mitinori Saitou

Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Hon-cho, Kawaguchi, Saitama, 332-0012, Japan

Laboratory of Molecular Cell Biology and Development, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan