Focus of the Month
Reverse Transcriptase: What's your choice?
To get an accurate and efficient yield of full length cDNA, one needs to make the right choice of the reverse transcriptase to be used.
The MMLV High Performance Reverse Transcriptase (MMLV HP RT) from Epicentre demonstrates significantly greater reverse transcriptase activity than other commercially available MMLV RT enzymes and also synthesizes full-length cDNA from RNA templates longer than 15 kb.
The test of a good reverse transcriptase is its capability to produce full length cDNA. The buffer provided with the enzyme from Epicentre has been optimized to produce full length cDNA as is evident from the 3’/5’ ratios observed (Refer to the table below for comparison of these ratios using reverse transcriptases of other well known companies).
Table 1. 3´/5´ ratio analysis of cDNA produced by different reverse transcriptase enzymes. Total cellular RNA from HeLa cells was converted to cDNA using the three reverse transcriptase enzymes indicated in the table. A 3´/5´ ratio equal to 1.0 means that equal amounts of PCR products are obtained from both the 3´ and 5´ end of the cDNA and therefore is a good indication that the reverse transcriptase has produced a full-length cDNA copy of the mRNA.
Another thing to make note of is the activity of RNase H. Reverse transcriptase enzymes with reduced RNase H activity produce more full length cDNA and provide greater gene product representation than enzymes with wild-type levels of RNase H activity. Epicentre’s MonsterScript™ Reverse Transcriptase is a thermostable reverse transcriptase that completely lacks RNase H activity. Its thermostability enables it to perform at temperatures greater than 50?C, thus overcoming problems due to secondary structure in longer transcripts. The enzyme's lack of RNase H activity contributes to its ability to make longer cDNAs and more complete full-length libraries of first-strand cDNA molecules compared to other reverse transcriptases. Epicenter scientists have demonstrated reverse transcription of 15-kb RNA templates using MonsterScript™ Reverse Transcriptase. The buffer contains betaine which reduces pausing and stopping of the transcriptase, enabling efficient reverse transcription of difficult and GC-rich templates.The kit includes a potent RNase Inhibitor to protect the integrity of template RNA. The long citation list speaks for itself.
- Liang, C.-Y., et al. (2011) The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1. Nucleic Acids Res. , gkr040.
- Vestergaard, A. L., et al. (2011) Transcriptional expression of type-I interferon response genes and stability of housekeeping genes in the human endometrium and endometriosis. Mol. Hum. Reprod. 17, 243-254.
- Wang, L.-C., et al. (2011) Involvement of the Arabidopsis HIT1/AtVPS53 tethering protein homologue in the acclimation of the plasma membrane to heat stress. J. Exp. Bot. , err060.
- Catalano, P. N., et al. (2010) Lack of functional GABAB receptors alters GnRH physiology and sexual dimorphic expression of GnRH and GAD-67 in the brain. Am J Physiol Endocrinol Metab 298, E683-696.
- Chen, T.-L., et al. (2010) Contribution of a Plasmid-Borne blaOXA-58 Gene with Its Hybrid Promoter Provided by IS1006 and an ISAba3-Like Element to {beta}-Lactam Resistance in Acinetobacter Genomic Species 13TU. Antimicrob. Agents Chemother. 54, 3107-3112.
- Malathi, K., et al. (2010) RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP. RNA 16, 2108-2119.
- Rajanbabu, V., et al. (2010) Tilapia Hepcidin 2-3 Peptide Modulates Lipopolysaccharide-induced Cytokines and Inhibits Tumor Necrosis Factor-{alpha} through Cyclooxygenase-2 and Phosphodiesterase 4D. J. Biol. Chem. 285, 30577-30586.
- Ramirez, M. C., et al. (2010) Differential neonatal testosterone imprinting of GH-dependent liver proteins and genes in female mice. J. Endocrinol. 207, 301-308.
- Tan, D. and Walker, A. M. (2010) Short form 1b human prolactin receptor down-regulates expression of the long form. J. Mol. Endocrinol. 44, 187-194.
- Tian, Y., et al. (2010) MicroRNA-10b Promotes Migration and Invasion through KLF4 in Human Esophageal Cancer Cell Lines. J. Biol. Chem. 285, 7986-7994.
- Wu, Z.-B., et al. (2010) Biological and Molecular Characterization of Apple chlorotic leaf spot virus Causing Chlorotic Leaf Spot on Pear (Pyrus pyrifolia) in Taiwan. HortScience 45, 1073-1078.
- Xin, Q.-Q., et al. (2010) Apoptosis contributes to testicular toxicity induced by two isomers of bromopropanes. Toxicology and Industrial Health 26, 513-524.
- Chen, G.-Y., et al. (2009) Baculovirus Transduction of Mesenchymal Stem Cells Triggers the Toll-Like Receptor 3 Pathway. J. Virol.83, 10548-10556.
- Bae, A. S., et al. (2011) Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples. J. Clin. Microbiol. 49, 3168-3174.
- Cheng, G., et al. (2011) Selection of Clinically-relevant Protease Inhibitor Resistant Viruses Using the Genotype 2a HCV Infection System. Antimicrob. Agents Chemother. , AAC.01382-10.
- Cheng, G., et al. (2011) Selection of Clinically Relevant Protease Inhibitor-Resistant Viruses Using the Genotype 2a Hepatitis C Virus Infection System. Antimicrob. Agents Chemother. 55, 2197-2205.
- Chen, W., et al. (2010) Disruption of ptLPD1 or ptLPD2, Genes That Encode Isoforms of the Plastidial Lipoamide Dehydrogenase, Confers Arsenate Hypersensitivity in Arabidopsis. Plant Physiology 153, 1385-1397.
- Hernandez-Diaz, I., et al. (2010) The Mineralocorticoid Receptor Is a Constitutive Nuclear Factor in Cardiomyocytes due to Hyperactive Nuclear Localization Signals. Endocrinology 151, 3888-3899.
- Teterina, N. L., et al. (2010) Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags. J. Virol. 84, 1477-1488.
- Friedman, S. D., et al. (2009) Gene Mapping and Phylogenetic Analysis of the Complete Genome from 30 Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae). J. Virol. 83, 11233-11243.
- Kent, T., et al. (2009) The 5' external transcribed spacer in mouse ribosomal RNA contains two cleavage sites. RNA 15, 14-20.
- Budde, P. P., et al. (2007) Characterization of a higBA Toxin-Antitoxin Locus in Vibrio cholerae. J. Bacteriol. 189, 491-500.
- Chumley, M. J., et al. (2007) EphB Receptors Regulate Stem/Progenitor Cell Proliferation, Migration, and Polarity during Hippocampal Neurogenesis. J. Neurosci. 27, 13481-13490.
- Alice, A. F., et al. (2006) Genetic and Transcriptional Analysis of the Siderophore Malleobactin Biosynthesis and Transport Genes in the Human Pathogen Burkholderia pseudomallei K96243. J. Bacteriol. 188, 1551-1566.
For those looking for a coupled reaction with PCR, we have the following RT-PCR kits:
MasterAmp™ High Fidelity RT-PCR Kit
- High-fidelity RT-PCR (i.e., low error rates).
- Amplification of full-length products from RNA templates up to ~6 kb (Fig. 1).
- Uses MasterAmp PCR Enhancer for better amplification of high-GC or other difficult templates
Figure 1. The MasterAmp™ High Fidelity RT-PCR Kit amplifies full-length products from RNA templates up to ~6 kb. RT-PCR products from Tobacco Mosaic Virus RNA with primers that amplify a 5.5-kb region of the viral RNA. Lane M, Kilobase ladder; lane 1, RT-PCR using the MasterAmp one-step protocol; lane 2, RT-PCR using MMLV-RT and Taq DNA polymerase with a standard two-step procedure; lane 3, RT-PCR using TthDNA polymerase.
MasterAmp™ RT-PCR Kit for High Sensitivity
- High-sensitivity RT-PCR from as little as 1 pg of total cellular RNA (Fig. 2).
- Outperforms similar products from other vendors.
- Thermostable RetroAmp™ RT DNA Polymerase enables RT to be performed at higher temperature, which minimizes RNA secondary structure.
- The MasterAmp PCR Enhancer technology increases RT-PCR sensitivity and specificity.
Figure 2. Human CGa was amplified from various amounts of total human placental RNA using MasterAmp™ RT-PCR Kit for High Sensitivity or kits from other manufacturers. All experiments were carried out strictly according to each manufacturer's protocols.
So what’s your choice?
Sibenzyme is a ISO 9001:2008 certified company and is one of the leading companies in the production of restriction and modifying enzymes used in molecular biology. The SE product line includes more than 200 enzymes, several perfect DNA ladders, high quality dNTPs and DNA preparations.
Sibenzyme supplies to many well known brands across the world including NEB, Takara, Fermentas, Invitrogen, Promega, Roche and many more. To know more click here
Its R & D is dedicated to the development of new restriction enzymes and they can boast of several publications which can be viewed here.
Some commonly used Restriction Enzymes
Restriction Enzyme |
Restriction site |
| BamH I | G^GATCC CCTAG^G |
| EcoR I | G^AATTC CTTAA^G |
| EcoR V | GAT^ATC CTA^TAG |
| Hind III | A^AGCTT TTCGA^A |
| Hinf I | G^ANTC CTNA^G |
| NdeI | CA^TATG GTAT^AC |
| XbaI | T^CTAGA AGATC^T |
| PstI | CTGCA^G G^ACGTC |
| XhoI | C^TCGAG GAGCT^C |
| KpnI | GGTAC^C C^CATGG |
And many many more...
Also check the special range of Enzymes, dNTPs and ladders.
Modifying Enzymes |
| Taq DNA Polymerase |
| T4 Polynucleotide Kinase |
| Alkaline phosphatase |
| Klenow |
| T4 DNA ligase |
| MMLV Reverse transcriptase |
| T7 RNA polymerase |
And many more...
Ladders |
| 1 kb ladder |
| 100bp ladder |
| Lambda HindIII |
| 50kb ladder |
And many more...
Try them out! You shall soon get hooked on! Available from our ready stock!
Biotech Desk launches a range of kits and reagents for teaching and training students in Biotechnology and Molecular Biology
Biotech Desk has partnered with Indigenese Biotechnology to bring to the market indigenously manufactured kits and reagents for training labs in molecular biology. Each kit or reagent has been rigorously tested for its quality, stability and performance. Every kit and reagent is very reasonably priced yet not compromising on quality.
Kits include all of the reagents and samples required for performing an experiment (excluding equipment). Educators would find these extremely useful to run their practical labs.
List of Teaching kits:
Catalogue No. |
Name of the kit |
No. of reactions |
| INDI T001 | Plasmid extraction kit | 10 |
| INDI T002 | Genomic DNA extraction kit | 10 |
| INDI T003 | Ligation kit | 5 |
| INDI T004 | Restriction Digestion kit | 5 |
| INDI T005 | Colony PCR kit | 10 |
| INDI T006 | PCR kit | 10 |
| INDI T007 | DNA amplification kit | 5 |
| INDI T008 | RFLP kit | 5 |
| INDI T009 | Agarose gel electrophoresis kit | 10 |
| INDI T010 | SDS-PAGE kit | 5 |
| INDI T011 | Transformation kit-1 | 5 |
| INDI T012 | Transformation kit-2 (with competent cells) | 5 |
List of Teaching kits:
Catalogue No. |
Name of the reagent |
Quantity |
| INDI S01 | 50X TAE | 100 ml |
| INDI S02 | 10x TBE | 100 ml |
| INDI S03 | 1M Tris pH 8.0 | 100 ml |
| INDI S04 | 1M Tris pH 7.5 | 100 ml |
| INDI S05 | 1M Tris 8.8 | 100 ml |
| INDI S06 | 1M Tris 6.8 | 100 ml |
| INDI S07 | 0.5 M EDTA pH 8.0 | 100 ml |
| INDI S08 | 10x TE | 100 ml |
| INDI S09 | 5x Tris-glycine buffer | 100 ml |
| INDI S10 | 3M Sodium acetate | 100 ml |
| INDI S11 | 10x DNA loading dye | 1 ml |
| INDI S12 | 10x SDS-PAGE loading dye | 1 ml |
| INDI E01-100 | New product: Taq polymerase | 100U |
| INDI E01-500 | Taq polymerase | 500U |
| INDI E01-1000 | Taq polymerase | 1000U |
| INDI E02-100 | New product: Pfu polymerase | 100U |
| INDI E02-500 | Pfu polymerase | 500U |
| INDI E02-1000 | Pfu polymerase | 1000U |
Assay ready Luc reporter constructs for over 20,000 genes.
The 3’-UTR sequence of a gene of interest was cloned downstream of the firefly luciferase gene. The chimeric transcript level is regulated by its interaction with miRNA(s), which results in varied luciferase activity quantifiable by a colorometric assay.
In addition a CMV promoted RFP gene is present in the plasmid to works as a transfection indication.
- SV40 is a strong promoter
- High sensitivity of Luciferase assay due to IRES driven Luciferase cassette
- RFP as a transfection indicator
- Neomycin selection marker for stable cell development.
- Origene provides genome wide 3’UTR clones for human
Working Mechanism:
What Origene offers:
- Ready-to-use vector with 3’UTR of a specific gene in the reporter vector
- Coverage: human genome, searchable by Accn or gene name
- Control vector is available for purchase but not included
- Deliverable: 10ug of purified plasmid, lyophilized in a 2-D barcoded vial.
To identify the clone of your choice visit: http://www.origene.com/MicroRNA/3-UTR-Clone/ or write to us at support@biotechdesk.com
FailSafe™ PCR System- Successful PCR, the first time and every time
Stop paying out extra on changing polymerase types for an unsuccessful PCR.
A complete solution to all PCR related problems without changing polymerase type. The FailSafe™ PCR System provides dependable, consistent high-fidelity PCR results for every DNA template, regardless of its source or sequence. The FailSafe PCR System will faithfully amplify your template every time.
Celebrating the 10 glorious years of FAILSAFE solving PCR problems for the customers.
WHY PCR WITH FAILSAFE?
- Simplified PCR optimization.
- PCR of difficult or high-GC templates (Fig. 1).
- Multiplex PCR (Fig. 2).
- PCR amplifications up to 20 kb for cloning.
Figure 1:
Amplification of an 80%-85% GC-rich region of the human fragile X gene. PCR was performed using the FailSafe™ PCR System, schematically depicted above. Lanes A-L show the amplification products resulting from PCR using the 12 FailSafe PCR PreMixes. Lane M, molecular weight marker. In this experiment, optimal amplification was obtained with FailSafe PCR PreMix J. The size of the expected amplicon is indicated by an arrow.
Figure 2:
Multiplex PCR of the human CFTR gene. The FailSafe™ PCR System amplified all five exons of the CFTR gene from as little as 1 ng of human genomic DNA. Lane M, DNA marker; lane 1, negative control; lanes 2-5, multplex PCR from 1, 10, 50, and 100 ng, respectively, of human genomic DNA.
Using EPICENTRE's patented PCR Enhancement Technology, the FailSafe PCR PreMix Selection Kit has replaced the need for cumbersome adjustments of reaction components to determine the optimal conditions for PCR of each template and primer pair. The 12 FailSafe PCR PreMixes in this kit cover a meticulously determined matrix of PCR conditions that give optimal PCR results for different sequences. Each PreMix represents a unique PCR condition, with everything needed for successful PCR. Combine the template and primers with the FailSafe Enzyme Mix, add one volume of this cocktail to one volume of each of the 12 FailSafe PCR 2X PreMixes, and cycle. EPICENTRE guarantees that at least one FailSafe PCR PreMix will provide conditions that are optimal for the template and primers tested
For reliable and consistent PCR results for all subsequent amplifications using the same primers, simply order the FailSafe PCR System with PreMix Choice, and specify the same FailSafe PCR PreMix that gave optimal PCR results using the FailSafe PreMix Selection Kit.
Benefits
- Successful PCR, the first time and every time.
- High PCR accuracy, using a unique PCR Enzyme Mix with a 3-fold lower error rate than Taq DNA polymerase.
- Extremely high sensitivity and specificity using the PCR Enhancer Technology.
- FailSafe PCR PreMixes contain all components needed for successful PCR.
- Generates PCR products suitable for both TA cloning and blunt-end cloning.
Citations
- Arnett, J., et al. (2011) Autosomal Dominant Progressive Sensorineural Hearing Loss Due to a Novel Mutation in the KCNQ4 Gene.Arch Otolaryngol Head Neck Surg 137 , 54-59.
- Felsner, G., et al. (2011) ERAD Components in Organisms with Complex Red Plastids Suggest Recruitment of a Preexisting Protein Transport Pathway for the Periplastid Membrane. Genome Biol Evol 3 , 140-150.
- Hamer, S. A., et al. (2011) Discovery of diverse Borrelia burgdorferi strains in a bird-tick cryptic cycle. Appl Envir Microbiol , AEM.02479-10.
- Noben-Trauth, K. & Latoche, J. R. (2011) Ectopic Mineralization in the Middle Ear and Chronic Otitis Media with Effusion Caused by RPL38 Deficiency in the Tail-short (Ts) Mouse. J Biol Chem 286 , 3079-3093.
- Aiello, D., et al. (2010) Discovery and Characterization of Inhibitors of Pseudomonas aeruginosa Type III Secretion. Antimicrob Agents Chemother , AAC.01598-09.
Time to go mad with our prices!! Limited period offer till March 31st 2010
The March Hare is visiting again. Huge discounts on our purification kits, enzymes and kits for RNA research. Enjoy the MadHatter’s tea party as long as it lasts! - Download page 1 | Download page 2
Focus of the Month: Metagenomics
A drop of cesspool water or the soil in your backyard may be an invaluable resource of new enzymes or bioactive compounds. A consortium of bacteria and fungi grow and thrive together and are oftentimes difficult to cultivate as a pure laboratory culture. The science of metagenomics addresses the collective genomic DNA of all these organisms which were not cultivable as separate entities.
Making Metagenomic Libraries:
First one needs to isolate high molecular weight intact DNA in sufficient quantity in order to make a successful metagenomic library. Researchers make either BAC or Fosmid libraries using BAC or Fosmid cloning vectors. Biotech Desk supplies Fosmid and BAC library construction kits from Epicentre Biotechnologies,USA. There are innumerable citations of the use of these kits for the construction of metagenomic libraries. In fact the Copy Control Fosmid Library construction kit is on the SOP of the JGI (Joint Genome Institute). You could also visit the blog on Epicentre’s website to read more about the latest developments in this area:
http://epicentral.blogspot.com/search/label/metagenomics
Isolation of Metagenomic DNA from Soil and other environmental samples:
Recently, Epicentre launched the Meta-G-Nome™ DNA Isolation Kit, which has made the task of DNA purification from diverse environmental sample types very easy. The kit generates ~40-kb DNA that can be used directly in construction of a metagenomic DNA library using the CopyControl™ Fosmid Library Production Kit. The isolated DNA is also well suited for PCR screening or next-generation sequencing. Usually researchers pick on the SoilMaster DNA extraction kit or the ExtractMaster DNA extraction kits for extraction of DNA from soil or feces. These kits are extremely good in purifying high quality DNA from the respective sources which are PCR-ready; however they should not be used for genomic DNA extraction with a purpose to build a library. The Metagenomic DNA Isolation Kit for Water helps isolate 40kb fragments of genomic DNA from microbes present in water samples.
Fosmid and BAC clones can be purified for sequencing and other downstream applications using the BACMAX™ DNA Purification Kit or the FosmidMAX™ DNA Purification Kit.
Biotech Desk has also recently established a partnership with Amplicon Express, USA who are the world leaders in library construction. Apart from the regular BAC libraries of plants and animals, they also specialize in construction of metagenomic libraries from soil, sludge, marine and other environmental samples.
Citations for the Copy Control Fosmid Library construction kit:
http://www.epibio.com/product_citations.asp?id=385
Citations for CopyControl™ BAC Cloning Kits (BamH I, EcoR I, or Hind III)
http://www.epibio.com/product_citations.asp?id=380
Citations for pWEB-TNC™ Cosmid Cloning Kit
http://www.epibio.com/product_citations.asp?id=332
You may also wish to buy the following related products:
- BAC-Tracker™ Supercoiled DNA Ladder
- Lambda Terminase
- TransforMax™ EPI300™ Electrocompetent E. coli
- TransforMax™ EPI300™ Chemically Competent E. coli
- TransforMax™ EPI300™-T1R Electrocompetent E. coli
- TransforMax™ EPI300™-T1R Chemically Competent E. coli
- CopyControl™ Fosmid Autoinduction Solution
- pCC2 Forward & Reverse Sequencing Primers
- pWEB-TNC™ Cosmid Cloning Kit
- pWEB™ Cosmid Cloning Kit
- Supercoiled DNA Marker Set
- Epi-Grids™ Colony Grid Templates