A: The tips fit most conventional single or multi-channel pipettors - manual or automatic. At this time, they are not compatible with the Rainin LTS model.
2.Q: What PCR sample volume can I purify using a single tip?
A: Currently, the tip is optimized for a 25ul PCR reaction sample.
3. Q: Do I have to use all 96 tips at once?
A: You do not have to use all 96 tips at once - you can use only as many as you need at a time. This makes our product very scalable & flexible if the number of PCR reaction samples change each time you run a PCR. The unused tips can be stored in the box on your shelf at room temperature.
4. Q: What is the shelf life of the tips?
A: 1 year
5. Q: Is the RapidTip compatible with TOPO cloning (one of the lesser DNA polymerase cloning techniques).
A: With TOPO cloning, the goal is to remove primers and primer-dimers after PCR prior to cloning. As the RapidTip removes nucleotides, primers, and primer-dimers < 45-50 bp it should work well for TOPO cloning.
6. Q: What is the reaction volume and how much liquid is lost inside the tip?
A: RapidTip is optimized for 25 microliter reactions but can tolerate 20-30 microliters with no modifications. Typically 5 microliters is lost inside the tip.
7. Q: Where in the purification pipeline do you use the RapidTip product?
A: Basically, you use the RapidTip for post-PCR purification - this is where you'd typically use a spin column, enzymatic, or magnetic kit for PCR purification. The RapidTip removes DNA impurities (nucleotides, primers, and primer-dimers < 50bp) from the PCR reaction while leaving the PCR amplicon (>100bp), salt, and polymerase in solution. Salt and polymerase do not interfere with downstream reactions such as Sanger sequencing in our extensive tests (salt and enzymes are also present in Exo-SAP treated PCR).
Below find an outline of a typical workflow where I've identified the right place for RapidTip purification (step 3 in bold).
1. DNA Extraction: from tails, tissue, etc. Purification may or may not be included in all protocols.
2. PCR amplification: use genomic DNA from 1. as template for PCR amplification of your region of interest.
3. RapidTip Purification: use the Diffinity RapidTip to remove dNTP, primer, and primer-dimer from your PCR reaction - amplicon will remain in solution.
4. BigDye Reaction: use RapidTip purified PCR amplicon from 3. as template for your sequencing reaction.
8. Q: Can the Diffinty RapidTip be used for PCR products 50-99 bp long? Most of my PCR products that I need to sequence are 60-100 bp long (I use real-time RT-PCR and design my own TaqMan primers and probes, all amplicons between 50 and 150 bp). I don't need 100% recovery, just enough to get ~10 µl with DNA concentration of at least 25 ng/µl for sequencing
A: The RapidTip hasn't been tested for sequencing PCR fragments in the 50-99bp range. It will remove some of the dsDNA between 50-99bp. At 100bp you can expect at least 80% yield and between 50-75bp you may see at least 50% yield and between 75 and 99bp perhaps 65% yield.
As you know, the final concentration will depend on your initial concentration. So if you had a 50 ng/uL initial concentration, it's likely you'd get 25 ng/uL in the 50-99 bp size range as you would obtain at least 50% yield. We recommend you check your DNA concentration via gel or nanodrop prior to sequencing.
9. Q: Will be testing to see if we can clean up probes for in situ hybridization to cells. Question: Will tip also retain single stranded large DNA (100-200 bp)?
A: Unfortunately, we haven’t tested retention of large single strands, so we don't know – but our initial answer is probably not. Most large single-stranded DNA hybridizes on itself at room temperature which would make it more like double stranded DNA between, say 50-100bp, which would mostly pass through, as our tip is optimized to retain dsDNA of 100bp and larger. Some would be retained - but you would need to determine if it was enough for your application
If you decide to test our RapidTips to see if they retain large ssDNA, please let me know how they worked for you or if you have any other questions, don’t hesitate to contact us.
10. Q: Do you have data on use of the RapidTips for library preparation for next-generation sequencing libraries? I currently use solid phase reversible immobilization (SPRI) magnetic beads for purification, and would like a faster/cheaper method for preparing large numbers of libraries in parallel.
A: At this time, we don't have data for preparation of next-gen sequencing libraries. All of our testing to date has been for purification prior to Sanger sequencing. However, we are seeking individuals that may be interested in testing our product for library preparation for next-gen sequencing and assisting us with defining performance features to ensure we are meeting the needs of our customers. Would you be interested in being a tester? If so, let us know, and I'll ensure someone contacts you.
11. Q: Can the Diffinity RapidTip be used to purify 15ul samples?
A: When you made your sample request, you had asked about the RapidTip’s ability to purify 15ul samples. We have not tested the performance of our tip for a sample of that size. We have optimized the tip to handle 25ul (20 -30ul) PCR samples for the best results from purification. Therefore, our best solution would be to ask that you run 25ul PCR samples.
However, when you receive our samples, you may want to try our product with your 15ul samples. There is a sample volume loss of about 5ul on a 20-30ul PCR sample, so we assume you can expect a similar volume loss on your 15ul sample size. Depending on your starting concentration, you may have enough for sequencing – but again, we haven’t tested it. Another suggestion (untested) is you could possibly elute up to 20ul using a 1x PCR buffer, then use our tip to purify - then you should potentially get anywhere from 50-70% yield.
If you decide to test them with your 15ul samples, please let me know how they worked for you or if you have any other questions.
