Frequently Asked Questions

faq
  • DNA Release/Extraction
    • Q.What is the difference between microLYSIS® and microLYSIS®-Plus?
      A.It is a little confusing to have two products so similarly named, but microLYSIS® was the original reagent which works fine for many cell types. It doesn’t work so well for tough cells (like plants) and so later we changed the original reagent somewhat to make it more powerful and came up with microLYSIS®-Plus, which works on nearly all cell types. At that time we still had many people happily using microLYSIS® so we couldn’t just substitute the new solution. We always ask new people to try microLYSIS®-Plus as it is much more versatile. Both products are priced identically.
      Q.In what instance would you recommend microLYSIS® over microLYSIS®-Plus?
      A.The only sample type we would do this for is blood. For all other sample/cell types we recommend microLYSIS®-Plus.
      Q.Does either microLYSIS® or microLYSIS®-Plus contain mucolytics?
      A.No
      Q.Can microLYSIS®-Plus lyse any cell type?
      A.Most cell types can be lysed with this reagent. For more detailed information, an extensive list can be found in the:
      Q.Is microLYSIS®-Plus suitable for lysing attached cells?
      A.This depends on where the cells are. If they are attached to the bottom of a microtitre plate they can be lysed, as long as the reagent covers the cell and the lysis protocol can be carried out. If the cells are attached to a petri dish the low volume microLYSIS®-Plus is not suitable. The cells should be lifted and pelleted.
      Q.What is the recommended number of cells to lyse with microLYSIS®-Plus?
      A.If you know how much DNA you require, it is possible to calculate the relevant number of cells, i.e. one human cell contains 6.6 pg of DNA (3 * 10e9 (the size of the human genome in bp) * 2 (because diploid) * 660 (mw of one bp) * 1.67 * 10-12 pg (weight of one Dalton) = 6.6 pg.
      Q.How should I scale the amount of reagent with the number of cells?
      A.For efficient lysis it is essential that the reagent completely covers the cells. Therefore our recommended lysis volume is suitable for most applications and is optimal for maximum efficiency. It is possible to scale the volume up or down to suit assays of greater or fewer cells, although this can affect the efficiency of the lysis.
      Q.Can I dilute microLYSIS® or microLYSIS®-Plus?
      A.This is not recommended, but sometimes (depending on the cell type and amount of DNA required) this may be possible.
      Q.Can I dilute the DNA afterwards?
      A.Yes, but make sure the diluent is nuclease free.
      Q.Are microLYSIS® and microLYSIS®-Plus suitable for Real-Time (quantitative) PCR?
      A.Yes.
      Q.Can microLYSIS® and microLYSIS®-Plus be used for RNA isolation?
      A.No, the severe heat steps destroy the RNA.
      Q.As microLYSIS® and microLYSIS®-Plus is an irritant, should it be opened in a microbiology safety cabinet?
      A.No, but this product should be handled only by those persons who have been trained in laboratory techniques and used in accordance with the principles of good laboratory practice.
      Q.What temperature should microLYSIS® be stored at?
      A.+ 4°C
      Q.What is the shelf life of microLYSIS®?
      A.4 years.
      Q.What temperature should microLYSIS®-Plus be stored at?
      A.We recommend storage at -20 °C, but it can safely be stored short term at +4 °C.
      Q.What is the shelf life of microLYSIS®-Plus?
      A.2 years.
      Q.Does storing either microLYSIS® and microLYSIS®-Plus at room temperature affect the efficiency of DNA release?
      A.We don’t recommend storing microLYSIS® and microLYSIS®-Plus at room temperature.
      Q.Do microLYSIS® and microLYSIS®-Plus inhibit PCR?
      A.microLYSIS® and microLYSIS®-Plus can make up 40% of most PCR mixtures without showing inhibition. The sample itself must contain very little PCR inhibitors. The amount of inhibitors allowed depends on the PCR system.
      Q.Do microLYSIS® and microLYSIS®-Plus shear DNA?
      A.No, the resulting DNA from microLYSIS® and microLYSIS®-Plus lysis will be completely intact.
      Q.How long are microLYSIS® and microLYSIS®-Plus samples stable for?
      A.If you need to store the lysed sample we recommend storage at -20 °C.
      Q.What is the difference between DNAMITE® Plant Kit 1 and 2?
      A.The difference between DNAMITE Plant Kit 1 and 2 is that Kit 2 contains an RNase A solution for cleavage of single-stranded RNA.
      Q.Which DNAMITE® Kit is suitable for genotyping mouse ear punches and tails?
      A.We recommend using microLYSIS®-Plus for these type of samples.
      Q.How much tissue should I use for the DNAMITE® tissue kit?
      A.Use between 0.5 to 1.0 cm2 of fresh or thawed tissue.
      Q.Does plant resin interfere with the DNAMITE® DNA extraction?
      A.No.
      Q.Can DNAMITE® for Difficult Cells DNA extraction kit be used to extract DNA from moulted feather samples?
      A.Yes.
      Q.Is the DNAMITE® Plant Kit suitable for samples that contain concentrations of polyphenol and sugar such as strawberry and grape?
      A.Yes.
      Q.Would the DNAMITE® Plant kit yield DNA suitable for AFLP’s molecular subtyping?
      A.Yes.
      Q.Is DNAMITE® DNA suitable for next generation sequencing?.
      A.Yes.
  • PCR
    • Q.What kind of polymerases are in the MegaMixes?
      A.MegaMix, MegaMix-Double, Blue MegaMix-Double and MegaMix-W contain a proprietary formula of non-hot-start Taq DNA polymerases. MegaMix-Gold and MegaMix-Royal contain a proprietary formula of hot-start Taq DNA polymerases.
      Q.What is the Magnesium concentration of the various MegaMixes?
      A.Our standard 1.1x mixes: MegaMix, MegaMix-W & MegaMix-Blue have a magnesium concentration of 2.65 mM; our standard 2x mixes: MegaMix-Double & MegaMix-Blue Double have a magnesium concentration of 5 mM (2.5 mM final concentration); and our high activity 2x mixes; MegaMix-Gold & MegaMix-Royal have a magnesium concentration of 6 mM (3 mM final concentration).
      Q.What is the pH of the MegaMixes?
      A.pH 9.0
      Q.What is the salt (KCl or NaCl) concentration of MegaMixes?
      A.None of our MegaMixes contain potassium chloride (KCl) or sodium chloride (NaCl). Each MegaMix is composed from a proprietary formula containing a designated concentration of ammonium sulphate (NH4)2SO4.
      Q.What concentration of Taq polymerase (units per microlitre) do the MegaMixes contain?
      A.Polymerase activity is determined by unit concentration and buffer composition. Therefore the unit concentration will not enable you to determine the activity and performance of our mixes and we suggest testing one of our mixes best suited to your assay to determine the performance. All our MegaMixes are ready-to-use PCR master mixes that have been optimised for high-end performance. Our range of non-hot-start mixes (MM, MMD, MMB, BMMD & MMW) are of a lower activity to our hot-start mixes (MMG & MMR). Of our hot-start mixes, MegaMix-Gold has been formulated to provide the highest activity of all our mixes and so is suited for particularly tricky and sensitive PCR assays.
      Q.Will the dye in MegaMix-Blue and MegaMix-Royal affect any downstream applications?
      A.No.
      Q.Does the dye reduce the activity and efficiency of the polymerase?
      A.Our ready loading dye has a negligible effect on the activity and efficiency of DNA polymerases.
      Q.Is the Taq in any of the MegaMixes capable of proof reading?
      A.None of our MegaMixes are capable of proof reading.
      Q.Do the MegaMixes stall at uracil residues and therefore can they be used on bisulfite-treated DNA?
      A.The MegaMixes are able to complement uracil residues during synthesis and are therefore suited for use on bisulfite-treated DNA.
      Q.Does the polymerase in MegaMix give blunt or sticky ended fragments?
      A.All our MegaMixes can generate products with 3’-dA overhangs with an incubation step of 10 mins at 70 °C post PCR.
      Q.What is the maximum fragment length the MegaMixes will amplify?
      A.The MegaMixes are highly efficient at amplifying DNA fragments up to 2 kb but depending on the specificity and efficiency of the primer they can amplify up to 3 kb.
      Q.What is the error rate for the MegaMixes?
      A.The MegaMixes have an average error rate of 2.2x10-5 errors per nucleotide per cycle, thereby producing an accuracy rate of 4.5x104.
      Q.For how long are the MegaMixes stable at -20 °C?
      A.This product is very stable and it can be freeze thawed many times. The MegaMixes have a three year expiry date - but will be stable for much longer in the freezer.
      Q.Is MegaMix-Gold appropriate for quantitative (real-time) PCR?
      A.Yes, MegaMix-Gold is well suited for quantitative (real-time) PCR assays and platforms.
      Q.Is MegaMix-Gold suitable for multiplex quantitative (real-time) PCR assays?
      A.Yes, MegaMix-Gold has been tested on sensitive and specific multiplex quantitative (real-time) PCR assays and is very well suited for this application.
      Q.Are the MegaMixes compatible with restriction enzymes?
      A.The activity of any restriction enzyme is dependent on salt concentration, magnesium concentration and pH. It varies as to how forgiving each restriction enzyme is to these factors and we cannot guarantee that a particular restriction enzyme will work in the MegaMix buffers. We suggest that if your sample quality is limited that you use microCLEAN to purify the DNA and then perform restriction digest with the recommended buffer and conditions supplied with the enzyme. Alternatively, if you have ample sample quality, then by all means try the restriction digest in one of the MegaMixes.
      Q.Which MegaMix would be best for optimising GC rich products?
      A.To enhance MegaMix-Gold and MegaMix-Royal for GC rich products, we recommend using our GC buffer:
      Q.Is it ok to add DMSO to MegaMix?
      A.Yes, but in very low concentration.
  • DNA Cleanup
    • Q.What is the difference between microCLEAN and the CA Solution?
      A.microCLEAN is optimised to clean-up small DNA fragments, whereas the CA Solution is optimised to clean-up large DNA fragments
      Q.What constitutes the “small DNA” suitable for microCLEAN?
      A.Sizes under 10 kb, e.g. PCR fragments, small plasmids & restriction fragments.
      Q.What constitutes the “large DNA” suitable for the CA Solution?
      A.Sizes over 10 kb, e.g. genomic DNA, BACs & large plasmids.
      Q.What is an appropriate tube size for microCLEAN?
      A.We recommend using either 0.2 ml or 0.5 ml microtube for microCLEAN clean-up.
      Q.What is an appropriate tube size for the CA Solution?
      A.We recommend using 1.5 ml microtube for CA solution clean-up.
      Q.What is an appropriate volume of TE?
      A.The appropriate volume of TE is dependent on the concentration of DNA you desire, bearing in mind that the volume of TE needs to completely cover the pellet. (Guide only: 10 µl for pellet in 0.2 ml microtube and 20 µl for pellet in 0.5 ml tube).
      Q.Should I use molecular grade water or TE?
      A.Molecular grade water is perfectly suitable for short term storage, whereas for long term storage we recommend using 10/1 TE.
      Q.If I do resuspend in water will the samples be suitable for long term storage?
      A.We feel that DNA is always best stored in 10/1 TE as it inhibits DNases. However, if you are confident that the water is free from DNases the DNA should be stable long term.
      Q.Does microCLEAN inactivate enzymes during clean-up?
      A.microCLEAN is the ideal way for cleaning up any DNA after enzyme treatment, as it denatures the enzymes without precipitatating them.
      Q.What are the minimum and maximum volumes that microCLEAN can be used for?
      A.Any volume between 20 µl and 100 µl.
      Q.What are the minimum and maximum volumes the CA Solution can be used for?
      A.Any volume between 100 µl to 500 µl.
      Q.If I can’t see a pellet, does that mean that microCLEAN hasn’t worked?
      A.You should not see a pellet when using microCLEAN.
      Q.Does microCLEAN remove primer-dimers?
      A.Yes.
      Q.Is a microCLEAN'ed PCR fragment suitable for sequencing?
      A.Yes.
      Q.Can microCLEAN clean-up DNA from agarose gel slices?
      A.No.
      Q.Will microCLEAN remove RNA?
      A.Yes, microCLEAN only pellets double-stranded DNA.
      Q.Can I use either microCLEAN or the CA Solution to concentrate or dilute DNA samples?
      A.Yes.
      Q.What is the shelf life of microCLEAN?
      A.3 years.
      Q.How long can DNA be stored in water for?
      A.We cannot guarantee any definite period of time for which DNA can be stored in water, as this is dependent on a number of factors. DNA is less susceptible to degradation by DNases when stored at -20° C but can be damaged by repeat freeze/thaw cycles, whereas DNA stored at +4° C is more susceptible to degradation by DNases. Therefore if you plan to store your sample at +4° C we recommend resuspending in 10/1 TE to inhibit any DNases.
      Q.How long can DNA be stored in 10/1 TE for?
      A.We cannot guarantee any definite period of time for which DNA can be stored in 10/1 TE, but samples have been known to be stable for a long time both at +4° C and -20° C.
      Q.Can microCLEAN be used to remove SDS?
      A.Yes.
      Q.Does EDTA interfere with microCLEAN?
      A.No, but too much EDTA may co-precipitate with the DNA.
      Q.Does microCLEAN leave trace amounts of salt after clean-up?
      A.microCLEAN will only pellet double-stranded DNA and therefore the final solution should only contain DNA and water or 10/1 TE.
      Q.Do microCLEAN and the CA Solution give high yield?
      A.Both microCLEAN and the CA Solution are highly efficient and will provide as high a yield possible in relation to the amount of DNA present in the sample.
      Q.Is microCLEAN suitable for purifying plasmids?
      A.Yes.
      Q.Is microCLEAN’ed DNA suitable for cell transfection?
      A.Yes.
      Q.Can microCLEAN be used to purify DNA for Chromatin Immunoprecipitation (ChIP) experiments?
      A.Yes.
      Q.Can I use microCLEAN after a dephosphorilation process?
      A.Yes.
  • Sequencing
    • Q.What is the difference between BetterBuffer and BetterBase?
      A.BetterBuffer is designed for use with the ABI Big Dye® v1.0 and v3.0 sequencing mixes, whereas BetterBase is designed for use with the ABI Big Dye® v1.1 and v3.1 sequencing mixes.
      Q.Is it possible to rescale BetterBuffer and BetterBase sequencing reactions?
      A.BetterBuffer and BetterBase come with recommended ratios. However in some instances, experimentation has shown it possible to rescale the sequencing reaction and therefore if you have enough template to experiment with by all means give it a go.
      Q.How long can BetterBuffer and BetterBase be stored at +4 °C for?
      A.We recommend storing both BetterBase and BetterBuffer at -20 °C, but they are equally fine to be stored at +4 °C for the short term.
      Q.Are BetterBuffer and BetterBase compatible with the Beckman Coulter CEQ8800 DNA Analysis System?
      A.Yes.
      Q.Which sequencing buffer is best suited for use with the ABI 3730 machine with ABI BigDye® v3.1?
      A.BetterBase
  • Quality Testing
    • Q.Are the primers in the HDNAOK! kit tissue specific?
      A.The HDNAOK! Kit is comprised of ubiquitous human primers that are found in every human cell and tissue and is therefore not tissue specific.
      Q.How sensitive is the OK! KIT?
      A.The OK! Kit is able to distinguish a temperature difference of 1 °C.