Reagent Preparation and Storage
How should I store MicroGEM reagents?
MicroGEM reagents are thermostable which means they store well at room temperature. However, for prolonged life we recommend you keep reagents in the fridge, or in a freezer at -20•C for longer-term storage.
How should I store extracted DNA?
The DNA produced by MicroGEM reagents is stable but there are a few safeguards for longer-term storage:
- Remove any residual tissue from the extract and transfer the DNA to a new tube. Be aware that MicroGEM 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.
Why does my extracted DNA look like a smear when I run a gel?
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 MicroGEM extracts may not be photogenic, but they work extremely well in PCR-based applications. If you want to look at the DNA on a gel, skip the 95•C heat step in the extraction procedure and add 1 µl of RNase A to the loading dye.
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 protocol, you will have enough DNA for many experiments with a small amount of sample and short incubation times.
Can I shorten incubation times?
The 95°C step can often be reduced to 2 minutes, and the 75°C step can be reduced to 5 minutes. There may be a reduction in yield, but if you will have enough extracts if only performing a few PCRs. It’s worthwhile to do a little optimisation for your specific substrate.
Can I use a water bath or oven for my incubation instead of a thermocycler?
These will work but be sure to consider ramping times – it may take a while for the inside of the tube to reach the right temperature. Also, water baths are liable to contaminate your sample so take extra care. A thermocycler or MicroGEM’s PDQeX Nucleic Acid Extractor is better.
Can I make up bulk master mixes and store them?
No, do not store the diluted enzyme for more than 2-3 hours. Like most proteins, the MicroGEM enzyme will slowly adhere to the plastic tube wall. With other enzymes, adhesion is reduced by adding BSA to the reagents but you can’t use a protein to stablize a proteinase. The MicroGEM buffers contain surfactants to reduce this effect, but because adhesion is dependent on the type of tube, it is difficult to predict what loss will occur. Use your master mix fresh for best results.
How does the MicroGEM extracted DNA compare with automated bead 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 incubation. These methods usually produce high yields and high purity, but they have distinct disadvantages. Multiple pipetting and wash steps can introduce contamination and human error. These methods produce huge amounts of plastic and hazardous waste, and generally take hours rather than MicroGEM minutes. Just think of the multiple pipette tips required for those multiple wash steps versus MicroGEM’s single-tube approach.
For downstream PCR applications, the yield and purity of conventional methods are often unnecessary. These methods often have chemical carryover into the eluted DNA. With the MicroGEM method, the carryover into the PCR assay is limited to cellular extracts and degraded proteins which rarely affect PCR.
Remember, robots for DNA extraction by column or beads can only perform that function! MicroGEM’s extractions can be done on any liquid handling robot that can also be used for other purposes.
PDQeX Nucleic Acid Extractor
What's the difference between the PDQeX and conventional thermocyclers?
MicroGEM’s PDQeX Nucleic acid extractors. It uses our powerful temperature-driven enzymes with our proprietary extractor cartridge and purification matrix. The PDQeX uses heat to activate the enzyme and shrink the inner chamber, forcing the extract through a heat-burstable valve and purification matrix, perfect for removing inhibitors. This single-tube approach produces DNA extracts suitable for genotyping, sequencing, and the detection of pathogens in significantly less time (minutes rather than hours) than conventional methods.
Watch a quick video at https://www.youtube.com/watch?v=AkIYrioKLnM&feature=youtu.be to see a brief demonstration of how the extraction tube works.
The sliding door on my PDQeX won't open.
If the device is moved with the sample drawer inside, the movement can sometimes cause the drawer to shift, blocking the door from opening. It’s easy to fix if you have a #2 crosshead (Phillips) screwdriver.
Rotate the PDQeX on a table to expose the underside of the system as shown in the image. Remove the screws highlighted in yellow. You may need to remove the screws holding the rubber feet in place.
With the four screws removed, you can now lift the door assembly free from the device. Remove the sample plate that was blocking the door.
Realign the door assembly with the front of the device and reattach the screws.