The expected concentration values of my sample are too high or too low. Which steps are recommended for troubleshooting?
Please clean your measurement head and lid with a fluff-free tissue moistened with water or 70% ethanol. To avoid thawing effects make sure, that the sample has reached room temperature and it was mixed accurately before the measurement. We recommend using purified samples to avoid disturbing signals from impurities that absorb also in the region of interest.
Make sure that your pipettes are calibrated and the required tips are used. Please check whether you have selected the correct application (e.g. dsDNA, RNA, oligo) for your sample type. Ensure that the sample volume applied to the instrument correlates to the necessary volume for the selected pathlength (min. 1 µL for dilution 15/0.67 mm pathlength and min. 0.3 µL dilution 140/0.07 mm pathlength). Note, the 0.07 mm pathlength (equivalent to dilution 140) should not be utilized for samples with a concentration of less than 420 ng/µL.
Are the concentration results automatically converted or do I need to do additional manual calculations?
Within “Nucleic Acids” and “Protein UV” methods as well as in the Concentration, Standard Curve, and Protein Assays applications, the concentration values are automatically calculated. The A260 or A280 values are also displayed within these applications. In all other methods, the absorbance values (normalized to 10 mm) are provided.
Is it necessary to perform a purification protocol for protein samples before protein UV measurements?
Yes, absorbance measurements are not specific for a particular type of protein. The presence of other molecules, that also absorb at 280 nm, will influence measurement results (for more information, please see the question below “My samples show high absorbance in the 200-300 nm range; what could be causing this?”). For crude cell extracts it is recommended to perform a colorimetric assay—e.g. Bradford or BCA.
Is it necessary to perform a purification protocol for nucleic acid samples before the measurement?
Yes, absorbance measurements are not specific for a special type of nucleic acids. The presence of other molecules that absorb also at 260 nm does have an influence on the results.
Is it sufficient to clean the surface of the measurement head and the lid with a fluff-free tissue?
Yes, it is recommended to clean the measurement window surfaces with a fluff-free tissue. If using a sticky or hard to clean sample type, a DI water moistened fluff-free tissue can be used. If needed, 70% ethanol or isopropanol can be utilized for cleaning as well.
Are there problems with using solvents or aggressive substances?
In general, the surfaces of the measurement head are very resistant. However, it is not recommended to utilize concentrated acids or bases on the instrument. For questions regarding specific solvent compatibility, please contact the Implen-GO support team . A list of compatible solvents can be found in the user manual as well as here.
Does the Implen software also work on my Mac?
Yes, the Implen software can be operated with Windows and Mac-based computers.
Does the NanoPhotometer® require a computer or notebook for operation?
The Implen-GO NanoPhotometer®-GO family is designed with the utmost flexibility. It can be either purchased with an integrated display as a stand-alone unit or controlled by a computer via USB. Control via smartphone or tablet is possible as well.
What is the ideal storage buffer for my nucleic acid samples?
DNA dissolves best at an alkaline pH; it should be stored in an alkaline buffer such as 10 mM Tris-HCl (pH 8.0 – 8.5) including DNase-free water to prevent acid hydrolysis. For long term storage, the addition of 1 mM EDTA is suggested to prevent nuclease activity; alternatively, sodium acetate-alcohol precipitate can be utilized. RNA should be stored under slightly acidic buffered conditions, (pH 4.0 – 5.0) to prevent nucleophilic self-attack. It is best to use fresh RNase-free water for buffer solutions and store at -80 °C.
Can I use ultra pure water as a buffer?
As the pH of water varies caused by the solvation of CO2 from air, it is not recommended to use (ultra) pure water as buffer alone.
Can storing DNA in buffers containing EDTA affect my downstream applications and will it affect my spectroscopic measurements?
In order to achieve optimal PCR results, EDTA should not be used in PCR buffers as it is a divalent chelator and will inhibit enzyme activity. Additionally, EDTA absorbs near 230 nm, which influences the 260/230 ratio of samples.
My samples show high absorbance in the 200-300 nm range; what could be causing this?
– Urea, EDTA, carbohydrates, phenol, Guanidine HCl, as well as peptide bonds, absorb between 200 and 230 nm.
– TRIzol absorbs at 230 and 270 nm
– Guanidine isothiocyanate absorbs at 260 nm
– High sample turbidity can be indicated by an absorbance present at 320 nm.
– Fatty acids absorb between 200 and 300 nm
– NDSBs absorb between 230 and 270 nm
– RIPA absorbs between 250 and 290 nm
– Triton X-100 absorbs between 250 and 300 nm
For more information see Technical Note – Protein
What steps are recommended to test for cross-contamination or residue left behind from previous samples?
After cleaning the measurement head, apply your blank solution and run it as a sample. If no concentration is detected (flat measurement line at zero) the measurement head is completely clean. If a concentration result is displayed, please repeat the cleaning process. It is recommended to use a tissue moistened with 70% ethanol to properly remove sticky or highly concentrated protein samples.
Which cuvettes can be used with the C40?
A cuvette of any kind (quartz, glass or plastic) from any manufacturer can be utilized. The following pathlength settings can be adjusted: 10, 5, 2, 1 and 0.5 mm. For microvolume cuvettes, a z-height of 8.5 mm is required for compatibility.
What is the correct orientation of the DiluCell™ cuvettes in the OD600?
The light path of the DiluPhotometer™ OD600 passes the cuvette in the up/down direction at a center height of 8.5 mm.
What volume is required to use the DiluCell™ cuvettes?
DiluCell™ 10 requires a volume of 300 µL.
Why am I getting fluctuating values with the DiluCell™?
First, check for air bubbles in the DiluCell™ and if the necessary correct pipetting technique to avoid bubbles. Also, ensure the DiluCell™ has a tight fit in the cuvette holder. If the DiluCell™ is free to wiggle, the tilted orientation may cause reflections that affect absorbance readings.
What volume is required to use the DiluCell™ cuvettes?
DiluCell™ 10 requires a volume of 300 µL.