For better DNA gel extraction results, minimize exposure to UV light as much as possible
UV light is notorious for damaging DNA. Cut your gel slice quickly. When working with multiple bands, trim one at a time on the UV as opposed to letting the UV light damage the bands that are waiting to be cut. Alternatively, use a more visible range stain instead of UV light.
The best way to determine the purity of my DNA?
The ratio of absorbance at 260 nm to the absorbance at 280 nm (A260/A280) is typically used to measure the purity of the sample. This is because DNA absorbs light most strongly at 260nm so the absorbance value at this wavelength can be used to estimate the DNA concentration using an equation derived from “Beer’s Law”. A ratio of ~1.8 is generally accepted as pure for DNA, whereas a ratio of ~2.0 is generally accepted as pure for RNA, however as the aromatic amino acids, tyrosine and tryptophan absorb strongly at 280 nm, a decrease in the ratio is used as an indicator of protein contamination.
The A260/A230 is another indicator of contaminants, the expected values are commonly in the range of 2.0-2.2, Contaminants such as EDTA, carbohydrates, phenols and have an absorbance close to 230 nm and so will decrease the ratio, whereas guanidine isothiocyanate will increase the ratio. A poorer ratio is therefore often found when extracting using nucleic acid purification columns, although this may not interfere with downstream processes.
Thank you, Tennille Sibbritt from the Children's Medical Research Institute - Embryology Unit, for a great review on our SensiFAST™ SYBR® No-ROX Kit.
"I am preparing samples for RNA-seq using a protocol that has been optimised for low input samples. I performed a qPCR on two housekeeping genes (Actb and Tbp) as a quality control step to ensure good amplification of these genes before proceeding to library preparation. I initially performed the qPCR using a combination of Taq and SYBR, which led to Ct values of 20-22 for Tbp and 23-26 for Actb. I repeated this qPCR using the 200 L sample of SensiFAST SYBR No ROX master mix I received. This worked very well and was quite sensitive. The Ct values for Tbp were 19-20 and for Actb were 17-19, while the no template control had minimal amplification. I plan on using SensiFAST SYBR No ROX master mix in the future, especially since it is substantially cheaper than what I was previously using."
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illumigene Malaria by Meridian Bioscience
The diagnosis of Malaria (Plasmodium sp.) has remained a critical piece in both the control of this vector borne disease and clinical case management of patients affected by malaria.
Traditional methods of diagnosis have long remained entrenched as the status quo. Methods such as microscopy and rapid diagnostic tests (RDTs) have continued to be used in both non-endemic and endemic settings. Molecular detection methods have emerged, but have so far fallen short of widespread adoption due to a number of key factors: bulky, specialist capital equipment requirements, expense, highly-trained staff with expertise, cold storage of reagents, complex workflows & impractical field use.
illumigene Malaria by Meridian Bioscience, www.meridianbioscience.eu in the space of 12 months has literally transformed molecular diagnosis offering an innovative choice of molecular diagnostic assays suited not only to non-endemic, Western European laboratories, handling imported malaria cases, but serving a crucial role in both the endemic regions of sub-Saharan Africa and the sub-microscopic, low parasitemia reservoirs of asymptomatic carriers - a known source of transmission. This latter group is key in the success or failure of any in-country elimination programme to achieve full eradication of malaria. An expert, namely, Professor Daouda Ndiaye, Dakar, Senegal has remarked in a Malaria World expert blog that “early data suggests it
illumigene, thus far, in the hands of a multitude of parasitology experts across Europe, Middle East and Africa, has repeatedly demonstrated its accuracy, with a proven negative predictive value (NPV) of 100% and equally it's performance in reliably screening for positives in less than 1 hour, achieving high levels of sensitivity and unparalleled ease-of-use.
During World Malaria Day, April 25, 2017, Dr Tom van Gool (Academic Medical Centre, Amsterdam, Netherlands) as part of a morning session on parasite diagnosis, will present ground breaking results from a multi-centric trial, following an evaluation of the illumigene Malaria assays. This scientific presentation will take place during ECCMID 2017 (The 27th European Congress of Clinical Microbiology and Infectious Diseases), www.eccmid2017.org hosted this year in Vienna, Austria; an event regarded by many as the leading flagship congress worldwide, for microbiologists and infectious disease specialists with a projected attendance of 15,000 participants and over 3,000 abstracts accepted as part of its broad scientific programme.
"illumigene Malaria will establish itself as a milestone in Malaria diagnosis history, much like the cell counter-blood analyser has done following its introduction into the routine laboratory."
"illumigene will set a new paradigm in reliably mitigating against false positives, thereby facilitating a far easier diagnosis."
"In situations where ILG calls a positive result as a frontline screen ahead of a haemoscopy negative result for the same blood sample(s), then further investigations will be required to reconcile the result with the clinical picture. For this reason, the result can be considered esoteric with clinical expertise and laboratory input being required."
Romualo Grande, MD
UOC Microbiologia Clinica
Sacco Ospedale, ITALY
#illumigene #MeridianBioscience #Malaria
ISOLATE II PCR and Gel Kit Trim the Gel Slice as Much as possible.
Remove as much excess gel as possible, in front of and behind the DNA. This may sound obvious but many people cut out a square around the gel, paying less attention to the front and back. The more you can remove encasing your DNA, the higher the yields.
Agarose gel electrophoresis is a common molecular biology technique used to separate DNA or RNA molecules by size. This is achieved by denaturing and applying a negative charge to our nucleic acid sample. The samples are then run through an agarose matrix with an electric field (Electrophoresis). Shorter molecules move faster and thus migrate further and in the gel over a given time period. Many factors are involved with regards to the final visualization step and achieving high resolution of the resulting bands on the agarose gel.
Tips to help improve resolution include: - Running the gel at a lower voltage for a longer period of time - Using a wider/thinner gel comb - Loading less DNA into each well
MangoTaq comes with a coloured reaction buffer that contains red and orange dyes, which separate during electrophoresis and provide quick reference points for monitoring the mobility of the DNA samples in the gel.