What is the difference between forensicGEM and prepGEM?
The difference lies in the formulations of the forensicGEM and prepGEM kits and the extra QC and validation that goes into making the forensicGEM products. Because most forensicGEM DNA extraction will be used with multiplex STR analysis kits such as Identifiler and Profiler, the forensicGEM buffers have been modified for these sensitive reaction conditions. With the prepGEM™ range, most of the downstream applications are more robust and so we can use more powerful buffers and additives. ForensicGEM™ also undergoes more tests for the presence of human DNA before being released for sale.
How should I store forensicGEM and prepGEM?
The prepGEM/forensicGEM enzyme is very thermostable which means that it stores well at room temperature. However, for prolonged life we recommend that you keep it in the fridge, or for longer-term storage place it in a freezer at -20°C. This is mainly to safeguard against the solution being contaminated once opened.
At ZyGEM we have seen no loss of activity when the enzyme was cycled from -20°C to + 20°C each day for a month. However, if you have a large tube, and you are going to use it many times, it is wise to split it into smaller portions.
How should I store forensicGEM/ prepGEM extracted DNA?
The DNA produced by prepGEM or forensicGEM is stable but given that the kits will be used on a range of substrates, a few commonsense safeguards should be taken for longer-term storage.
- Remove any residual tissue from the extract and transfer the DNA to a new tube. Be aware though that ZyGEM DNA is very high molecular weight and so centrifugation at high speeds will drop the DNA out of solution.
- Store at -20°C in a freezer.
- Buffer the DNA to 1x TE.
How do I quantify forensicGEM / prepGEM extracted DNA?
Because of the heat cycle DNA produced by ZyGEM kits is approximate 90% single-stranded and so fluorescence based methods using double-stranded specific dyes like PicoGEM (Invitrogen) will underestimate DNA yields. See our Application Note to advise on quantification.
The DNA I extracted from blood is pink. Will it work?
Yes. The extraction buffer is designed to precipitate most of the inhibitors. A small amount of inhibition remains but if you use the recommended amount of extract for you PCR (1 µl in a 25 µl reaction) you will not see inhibition. If for some reason, you need to use more DNA and inhibition becomes a problem, then we recommend using a heam-resistant Taq.
When I run my forensicGEM / prepGEM extracted DNA on a gel, it looks like a smear.
This is because the DNA is denatured during the heat step and also contains RNA. Hence, it does not run as a discrete band on a stained agarose gel. The ZyGEM extracted DNA is not photogenic, but it works exceptionally well in PCR-based applications for which the method is intended. If you do want to look at the DNA on a gel, miss out the 95°C step in the extraction procedure and add 1 µl of RNAse A to the loading dye.
Other kits give purer DNA. Surely these are better methods?
Ask yourself if you need pure DNA. If you are making gene libraries, performing genomic digests, or cloning, then we do not recommend the ZyGEM kits. However, PCR-based applications do not require polished DNA. What is the point of spending time and money removing all of the protein from your DNA when you add BSA to your PCRs? If you don't believe that the ZyGEM kits work - and work well - send us an email and we will give you a test kit.
Can I use ZyGEM extracted DNA for gene cloning?
If the process involves a PCR (amplicon libraries) then ZyGEM extracted DNA will work fine. If you want genomic DNA for preparing libraries, then we recommend using other methods.
When I digest tissue, there is a residue left. Should I increase my incubation time?
The aim is to extract sufficient DNA, not to create a peptide soup. Over digestion of tissue samples will increase your DNA yield, but it will also increase the level of inhibition. If you follow the ZyGEM methods, you will have enough DNA for many experiments with a small amount of tissue and short incubation times. Don't become fixated on the idea that you have to get all the DNA out. If you only need enough DNA for a few PCRs, why bother trying to get enough for 10,000 reactions rather than 1000?
Can I shorten the incubation times?
Almost certainly. The 95°C step can often be reduced to 2 minutes, and the 75°C step to 5 minutes. There may be a reduction in yield, but if you are intended to perform just a few PCRs, then you will have enough. A little optimisation of the method is worthwhile given that we cannot match a single method for the very many different types of substrates our customers use.
Can I use water baths or oven for my incubation instead of a thermal cycler?
These will work but be sure to consider ramping times - it may take a while for the inside of the tube to reach temperature. Also, water baths are liable to contaminate your samples so you will need to take a lot of care. A thermocycler or Peltier block is better.
Can I make up bulk mastermixes and store these?
No. Do not store the diluted enzyme for more that 2-3 hours. Like most proteins, the ZyGEM enzyme will slowly adhere to the plastic tube walls and the ZyGEM proteinase is quite hydrophobic. With other enzymes, adhesion is reduced by adding BSA to the reagents but of course you cannot use a protein to stabilise a proteinase. The ZyGEM buffers contain surfactants to reduce this effect, but because the level of adhesion is dependent on the type of tube you use, it is difficult to predict what loss will be experienced … use your mastermixes fresh for best results.
I had extracted trace samples and when I came back to them, the DNA had disappeared.
Look at the FAQ on DNA storage but another possible cause is adhesion of the DNA to the tube walls. This is a problem during storage of low concentration DNA samples produced by any method. Special tubes can be bought which reduce this effect and you can also add carrier DNA and BSA to the stored sample. Do a Google search – many sites talk about this problem.
What automation system do you recommend?
One of the goals of developing the ZyGEM extraction system, was to create a method that can be automated on most, off-the-shelf liquid handling robots. You do not need an expensive machine to use our methods.
How does ZyGEM extracted DNA compare with automated bead based methods?
Bead based purification methods are, like column based methods, generally dependent on the use of chaotropic salts and organic solvents, involving multiple wash steps and incubations. These methods generally produce high yield, high purity DNA, however they are significantly more costly than ZyGEM extraction methods in terms of: time, pipette tip usage: cost of the kit and cost of the robot.
For downstream PCR applications the yield and purity these methods generate is often unnecessary. One of the challenges of automating purification methods is the incidence of chemical carry-over into the eluted DNA. Whilst ZyGEM extraction methods do not yield pure DNA, the carry-over into the PCR assay is limited to cellular extracts and degraded proteins which rarely effect PCR.
Remember also, robots for DNA extraction by column or beads can do only that! ZyGEM allows you to use any liquid handling robot that can be used for other purposes.
Do I need to purify DNA?
This depends entirely on the downstream application. For PCR / qPCR based applications there is be no need to generate highly pure DNA. The ease of contamination of PCR reactions actually makes purification steps a negative. Our customers routinely use ZyGEM extracted DNA for tricky downstream assays, such as 40 to 60 plex PCR amplifications.
How Do I get Technical Support?
Technical support is available at firstname.lastname@example.org
Is ZyGEM extracted DNA suitable for quantitative PCR?
Yes, ZyGEM extracted DNA should be suitable for qPCR analysis using either Sybr Green™ or probe based methods. Quantitative PCR can also be used to accurately assess the quantity of DNA present in a sample, for example using Quantifiler™ for human DNA.
What yield of DNA should I expect from my ZyGEM extraction?
It will vary between sample types. Where possible, information is given in the technical guide but in general, enough DNA will be released for between 100 and 1000 PCRs.
What is the composition of the buffers used for ZyGEM extraction?
The composition of the buffers is proprietary information. However each buffer has been carefully optimised to suit typical downstream applications for the kit type – for example, all of the forensicGEM product range produce DNA suitable for the more widely used forensic profiling kits (for example Identifiler).