FREQUENTLY ASKED QUESTIONS
- What is the difference between forensicGEM™ and prepGEM™?
- How should I store forensicGEM™ and prepGEM™?
- How should I store forensicGEM™ / prepGEM™ extracted DNA?
- How do I quantify forensicGEM™ / prepGEM™ extracted DNA?
- When I run my forensicGEM™ / prepGEM™ extracted DNA on a gel, it looks like a smear.
- Other methods give much purer DNA. Surely these are better methods?
- Can I use forensicGEM™ / prepGEM™ extracted DNA for gene cloning?
- When I digest tissue/hair/insects there is a large amount of residue left. Should I increase my incubation time?
- Can I shorten the incubation times?
- Can I use waterbaths or oven for my incubation?
- Can I make up bulk mastermixes and store these?
- I had some trace samples that I extracted and when I came back to them, the DNA had disappeared.
- What automation system do you recommend?
- How does prepGEM extracted DNA compare with automated bead based methods?
- Do I need to purify DNA?
- How Do I get Technical Support?
- Is ZyGEM extracted DNA suitable for quantitative PCR?
- What yield of DNA should I expect from my ZyGEM extraction?
- What is the composition of the buffers used for ZyGEM extraction?
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1. 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 large multiplex STR analysis kits such as Identifiler and Profiler, the forensicGEM™ buffers have to be suited 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 substantially more tests at a major forensic laboratory before being released for sale. (back to top)
2. How should I store forensicGEM™ and prepGEM™?
The prepGEM™/forensicGEM™ enzyme is thermophilic and thermostable which means that it that 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. 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. (back to top)
3. How should I store forensicGEM™ / prepGEM™ extracted DNA?
The DNA produced by prepGEM™ or forensicGEM™ is largely single stranded and if mistreated, fragmentation can occur over time. A few commonsense safeguards should be taken for longer-term storage.
- Remove any residual tissue from the extract. If necessary, centrifuge the tube and transfer the DNA to a new tube
- Store at -20°C in a freezer without a frost-free cycle
- Buffer the DNA to 1x TE by adding 10x or 100x TE to the extract. (back to top)
4. How do I quantify forensicGEM™ / prepGEM™ extracted DNA?
Because of the heat cycle DNA produced by prepGEM™ or forensicGEM™ is approximate 90% single-stranded and so fluorescence based methods using double-stranded specific dyes like PicoGEM (Invitrogen) will underestimate DNA yields be a factor of approximately 8. Alternative methods are available. (back to top)
5. 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 so it does not run as a discrete band on a stained agarose gel. Also, the prepGEM™ /forensicGEM™ enzyme releases large amount of RNA as well as DNA, hence, prepGEM™ /forensicGEM™ extracted DNA is not photogenic, but it works exceptionally well in PCR-based applications for which the method is intended. (back to top)
6. Other methods give much 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 method. 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? (back to top)
Can I use forensicGEM™ / prepGEM™ extracted DNA for gene cloning?
Yes, any PCR based protocol should be compatible with ZyGEM extracted DNA.(back to top)
8. When I digest tissue/hair/insects there is a large amount of 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. 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 reaction rather than 1000? (back to top)
9. Can I shorten the incubation times?
Almost certainly. The 95°C step should not be reduced below 5 minutes, but you can reduce the 75°C step. There will be a reduction in yield, but if you are intended to perform just a few PCRs, then you will have enough with a 5 minute incubation. (back to top)
10. Can I use water baths or oven for my incubation?
These will work but be sure to consider ramping times and water baths are liable to contaminate your samples. A thermocycler or Peltier block is better. (back to top)
11. 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. With other enzymes, this is reduced by adding BSA to the reagents but of course you cannot use this with 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.
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12. I had some trace samples that I extracted and when I came back to them, the DNA had disappeared.
Look at the FAQ on DNA storage but another serious consideration is adhesion of the DNA to the tube walls. This is a problem during storage of low conctration 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 issue. (back to top)
13. 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, however more on the automation of ZyGEM technology can be found at www.zygem.com/automation. (back to top)
14. How does prepGEM 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 do generally produce high yield, high purity DNA, however they are significantly more costly than ZyGEM extraction methods in terms of time, pipette tip usage and cost of the kit. For downstream PCR applications the yield and purity these methods generate is often redundant and wasteful. 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. At high levels these can cause PCR inhibition, however the release of these compounds can be controlled by the duration of the initial incubation, see above. (back to top)
15. Do I need to purify DNA?
This depends entirely on the downstream application. For PCR based applications there should be no need to generate highly pure DNA. The ease of contamination of PCR reactions by a sneeze or cough underscores this. Our customers routinely use ZyGEM extracted DNA for demanding downstream assays, such as 40 to 60 plex PCR amplifications, or with ABI’s AmpFISTR Indentifiler. It would appear that it is more beneficial to amplify from single stranded DNA, than from high-purity double stranded DNA. (back to top)
16. How Do I get Technical Support?
Technical support is available. Click here to go our support page. (back to top)
17. 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. (back to top)
18. 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. (back to top)
19. 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). (back to top)