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Notes on handling DNA

  Notes on handling DNA
Nature of DNA  
  The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation. Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments, high temperature, and UV irradiation. Careful isolation and handling of high molecular weight DNA is necessary to ensure that it can be used well in various downstream applications. Damaged DNA could perform poorly in applications such as genomic Southern blotting, long-template PCR, and construction of cosmid libraries.
Handling fresh and stored material before the extraction of DNA  
  For the isolation of genomic DNA from cells or tissues, use either fresh samples or samples that have been rapidly frozen in liquid nitrogen and stored at - 70°C. This procedure minimizes degradation of crude DNA by limiting the activity of endogenous nucleases.
Storage of DNA  
  Store genomic DNA at +2 to +8°C. Storing genomic DNA at -15 to -25°C can cause shearing of DNA, particularly if the DNA is exposed to repeated freeze-thaw cycles. Plasmid DNA and other small circular DNAs can be stored at +2 to +8°C or at -15 to -25°C.
Drying, dissolving and pipetting DNA  
  Avoid overdrying genomic DNA after ethanol precipitation. It is better to let it air dry than to use a vacuum, although vacuum drying can be used with caution. Plasmid DNA and other small circular DNAs can be vacuum-dried.
To help dissolve the DNA, carefully invert the tubes several times after adding buffer and tap the tube gently on the side. Alternatively let the DNA stand in buffer overnight at +2 to +8°C. Minimize vortexing of genomic DNA since this can cause shearing.
Avoid vigorous pipetting. Pipetting genomic DNA through narrow tip openings causes shearing or nicking. One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA. Regular pipette tips pose no problem for plasmid DNA and other small fragments.
Determination of concentration, yield, and purity of DNA  
  DNA yields are determined from the concentration of DNA in the eluate, measured by absorbance at 260 nm. Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. Pure DNA has an A260/A280 ratio of 1.7–2.1. Absorbance readings at 260 nm should be between 0.1 and 1.0. Sample dilution should be adjusted accordingly. Use elution buffer or water (as appropriate) to dilute samples and to calibrate the spectrophotometer. Measure the absorbance at 260 and 280 nm, or scan absorbance from 220–320 nm (a scan will show if there are other factors affecting absorbance at 260 nm).

Notes on handling RNA

  Notes on handling RNA
Nature of RNA  
  RNA is far less stable than DNA. It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases, which are permanently present everywhere in the lab. To achieve satisfactory qualitative and quantitative results in RNA preparations, contaminations with exogenous RNases must be reduced as much as possible Avoid handling bacterial cultures, cell cultures or other biological sources of RNases in the same lab where the RNA purification is to be carried out.
Handling of RNA  
  All glassware should be treated before use to ensure that it is RNase-free. Glassware should be cleaned with detergent, thoroughly rinsed and oven baked at 240 °C for four or more hours before use. Autoclaving alone will not completely inactivate many RNases. Oven baking will both inactivate RNases and ensure that no other nucleic acids (such as Plasmid DNA) are present on the surface of the glassware. You can also clean glassware with 0.1% DEPC (diethyl pyrocarbonate). The glassware must stand 12 hours at 37 °C and then autoclave or heat to 100 °C for 15 min to remove residual DEPC.
Storage of RNA  
  Purified RNA can be stored at -80°C and is stable for several years at this condition.
Quantification of RNA  
  The concentration of RNA should be determinate by measuring the absorbance at 260 nm (A260) in a spectrophotometer. Readings should be greater than 0.10 to ensure significance. An absorbance of 1 unit at 260 nm corresponds to 40 μg of RNA per ml. This relation is valid only for measurements at neutral pH. The ratio between absorbance values at 260 nm and 280 nm gives an estimate of RNA purity (see below).
When measuring RNA samples, make sure that cuvettes are RNase-free, esp. if the RNA is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with 0.1 NaOH, 1 mM EDTA followed by washing with RNase-free water. Use the buffer in which the RNA is diluted for calibration of the spectrophotometer.
Purity of RNA  
  The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA with respect to the contaminants that absorb in the UV, such as protein. However, the A260/A280 ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH results in lower A260/A280 ratio and reduced sensitivity to protein contaminations. For accurate values, it is recommended to measure absorbance in 10 mM Tris Cl, pH 7.5. Pure RNA has an A260/A280 ratio of 1.9-2.1 in 10 mM Tris/HCl pH 7.5. Always be sure to calibrate the spectrophotometer with the same solution.
For determination of the RNA concentration, however, it is recommend diluting the sample in a buffer with neutral pH since the relationship between absorbance and concentration (A260 reading of 1 = 40 μg/ml of RNA) is based on an extinction coefficient calculated for RNA at neutral pH.

General FAQ

  General FAQ
What is the composition of the Elution Buffer?  
  Elution Buffer (M): 10 mM Tris pH 8.5 - 9
Elution Buffer BS: 10 mM Tris pH 8.5
Elution Buffer R: RNAse free water
All elution buffers may be replaced by DNase/RNase free water.
What is the DNA fragment size that can be obtained using Invisorb® Spin column kits?  
  The average size of DNA fragments is 20 – 30 kb, the maximum fragment size can be up to 50 kb. The average fragment size is in general suitable for all common analytic applications like PCR techniques, hybridisation and NGS.
What is the DNA fragment size that can be obtained using InviMag® kits for extraction with magnetic beads?  
  The average size of DNA fragments is 100-300 kb. The large fragments obtained are in general suitable for all common analytic applications like PCR techniques, hybridisation and NGS.
What can I do if the amount of the extracted DNA is too low?  
  1. Insufficient lysis: you can try to increase the lysis time and/or decrease the amount of starting material.
2. Insufficient mixing with the binding buffer: vortex several times or pipette sample up and down for mixing before transferring it to the spin column.
3. Incomplete elution of DNA: extend the incubation time with Elution Buffer to 5-10 min or repeat the elution step.
What are the effects of changing elution volumes?  
  The optimum yield is achieved by the volumes of the elution buffer specified in the respective manual. Smaller elution volumes result in higher concentrations, but lower yield. A higher elution volume (partially) increases the yield by a few percent, but the concentration is lower.
What is the cause of clogging of Spin Filter membranes, how can it be avoided?  
  In most cases, too much sample material was used. Please ensure that you use the amount of sample material specified in the respective instructions. You can also try to increase the centrifugation speed.
Another reason could be insufficient lysis of the starting material. In this case, increase the lysis time or use more lytic enzyme such as Proteinase K, Proteinase S or Lysozyme, depending on the protocol.
What can I do when the surface of the spin filter turns yellow?  
  This problem may be caused by incomplete lysis (see above to solve this problem) or by an insufficient wash step. Please add another 800 µl of Wash Buffer II to the Spin Filter, centrifuge at 13.000 g for 1 min and then discard the filtrate.
How do I perform a DNA precipitation to concentrate my sample?  
  Add 1/10 volume of 3M NaAcetate pH 5.2 and 2 - 2.5 volumes of ice cold 100% ethanol. Mix and store at -20°C for >1 hr to precipitate the DNA. Recover the DNA by centrifugation at full speed in a microcentrifuge for 15-20 min. Pour off the ethanol and wash the pellet twice with 70% ethanol at room temperature. Allow the pellet to air dry and resuspend the DNA in an appropriate volume of TE buffer or water.
What are the main causes for inhibition of downstream applications (for instance PCR reactions)?  
  This is mostly caused by ethanol residues that are removed after the last wash step in the drying phase. Please make sure that the membrane is completely dry before eluting the DNA (as mentioned in the protocol).
Another reason could be salt carryover during the elution of the DNA. To avoid this, please make sure that the wash buffer has room temperature and check it for salt precipitates. Precipitates can be dissolved by carefully warming.

Microbiome

  FAQs
How long can samples be stored in the Stool Collection Tube with DNA stabilizer?  
  Samples can be stored for up to 3 months at room temperature, which is defined as a temperature range of 15-30°C.
If samples are frozen at -80°C, they can be stored indefinitely.
How much stool sample needs to be collected?  
  The required sample quantity is 1 g. The stool collection tube with DNA Stabilizer includes a spoon for sample collection. If a spoonful of sample is taken, this automatically corresponds to the optimal sample volume of 1 g.
What is the amount of buffer in the Collection Tube with DNA Stabilizer?  
  The Stool Collection Tube contains 8 ml of DNA Stabilizer.
Can I use the Invitek products for stool collection and extraction for animal samples?  
  Yes, the Stool DNA Stabilizer as well as the PSP® Spin Stool Basic Kit for sample extraction have been used for animal and veterinary studies. Products have been used for a variety of animals like cattle, goat, or bears.
For which downstream applications are samples extracted with the PSP® Spin Stool Basic Kit or the InviMag® Stool DNA Kit suitable?  
  The PSP® Spin Stool Basic Kit as well as the InviMag® Stool DNA Kit yield high-quality DNA through the sample-specific purification process. The DNA can be easily used for all modern downstream applications such as multiplex qPCR or NGS.

Infectious diseases

  FAQs
What is the advantage of the RTP® Pathogen kit?  
  This kit has a unique buffer that is lyophilized in the extraction tube. The lyophilized buffer contains: salts, detergents, Carrier RNA and Proteinase K, allowing a 1-step lysis of the sample saving 60% of pipetting steps compared to conventional kits.
Is it possible to purify genomic or bacterial or viral DNA/RNA separately from the same sample when using the RTP® Pathogen or Invisorb®/InviMag® Universal Kit?  
  When different types of nucleic acids are present in a sample, you will get a co-purification of all of them. This means for example, if you are looking for a special viral nucleic acid in a human or animal sample you will also co-purify the genomic nucleic acid as well.

Genetic testing

  FAQs
What is the amount of sample I can use with the Invisorb® Spin Blood Mini Kit?  
  For DNA extractions you can use whole blood and blood stains (fresh, frozen, old or dry blood and buffy coat) in the range from
1 to 200 µl.
What is the average yield of DNA I can expect with the Invisorb® Spin Blood Mini Kit?  
  The average yield of highly purified DNA depends on the amount and specific characteristics of the starting material and will be up to 15 µg.
How should I thaw frozen blood sample tubes?  
  Frozen blood should be thawed in a 37°C water bath for 10–15 min, inverted carefully for several times and stored on ice until processing. Do not incubate longer on 37°C as necessary for thawing!

Life sciences

  FAQs
What is the amount of sample I can use with the Invisorb® Spin Plant Mini Kit?  
  For DNA extractions you can use up to 100 mg of a fresh or frozen plant species, for dried plant material 60 mg are recommended.
What is the average yield of DNA for the Invisorb® Spin Plant Mini Kit?  
  Due to the highly variable DNA content of different plants and plant materials we cannot give a generally valid measuring range. The average yield of highly purified DNA depends on the amount and kind of starting material and the lysis efficiency and can be up to
50 µg.
Can I use the Invisorb® Spin Plant Mini Kit also for DNA purifications from organisms such as algae, bryophytes, or fungi?  
  Yes, you can. Using the Invisorb® Spin Plant Mini Kit, the extraction of DNA has been performed successfully from species like Fucus vesiculosus (algae), Sphagnum sp. (bryophyta), Xerocomus badicus (fungi) and Cladonia sp. (lichens). Also here grinding of the starting material under liquid nitrogen the basis for a high yield.

Oncology

  FAQs
What is the sample amount to use with the Invisorb® Spin Tissue Mini Kit?  
  For DNA extractions you can use 0.5 – 40 mg tissue sample, up to 1.0 cm mouse tail, up to 0.5 cm rat tail, 10-106 eukaryotic cells.
What is the average yield of DNA using the the Invisorb® Spin Tissue Mini Kit?  
  Up to 50 µg can be obtained, depending on the amount and type of tissue sample. For cell culture max. 6 µg can be obtained from human cells and max. 10 µg from animal cells.

Smarter Nucleic Acid Sample Preparation