Hi Quinso,

I have worked with a lot of challenged samples - including archaeological remains and arthropods.

First, a few questions/considerations:

1. How are the bees preserved? (Dried, immersed in alcohol, fixed in formalin, etc.)

2. Did you grind up the bee prior to extraction? If so, how?

3. What method did you use to quantify the DNA? (spectrophotometer, fluorometer, gel quantification, real-time PCR, etc.)

Answers to these questions will help us troubleshoot.

Next, some suggestions:

4. If you still have DNA extract left over from your first round of bee extractions, you can clean it up and concentrate it with a small spin column like the one in the Qiagen MinElute PCR purification kit. These silica-based columns will help get rid of inhibitors and allow you to elute your DNA in very small volumes. I would start with this and see how it goes.

5. The Quick-gDNA kit is made for mammalian cells. Your bees are high in mucopolysaccharides because of their exoskeletons. These compounds are serious PCR inhibitors and are often co-extracted with DNA. If you want to try a whole new round of extractions, look for a kit like a mollusk DNA extraction kit that specifically deals with this problem. E.N.Z.A. Mollusc DNA Isolation Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) is a good one.

Good luck and feel free to follow up,

Meredith

Hi Qiniso, Facing such a situation, in our lab we use forensic procedures, which enable the recovery of DNA from about every kind of materials. We still have home-made kits, but I bet commarcial kits are available. That said, the concentrations that you obtained are not that bad. The problem is certainly the poor quality of DNA, which must be too degraded for PCR amplification.

Given that your material seems very precious, you could try to reamplify it, either with a kit like Genomiphi or with kits like the ones developed by NuGene which might be relevant for your samples.

Good luck, Sébastien

Hi Qiniso,

I have not worked with museum samples. But have worked with skeletal remains, not a good source of DNA especially 30 years old. I would agree with Sebastien. A commercial kit is likely the best way to go. I would not expect a good yield from samples 30 years old since there will be some degradation. Genomiphi might work. However, I am not certain if it will work as well with degraded DNA samples. You may need to try it to find out. You also may need to work on PCR optimization to get any amplification. You only need a little amplification to allow re-amplification of the PCR product. Good luck.

Hi Quinso,

I have worked with a lot of challenged samples - including archaeological remains and arthropods.

First, a few questions/considerations:

1. How are the bees preserved? (Dried, immersed in alcohol, fixed in formalin, etc.)

2. Did you grind up the bee prior to extraction? If so, how?

3. What method did you use to quantify the DNA? (spectrophotometer, fluorometer, gel quantification, real-time PCR, etc.)

Answers to these questions will help us troubleshoot.

Next, some suggestions:

4. If you still have DNA extract left over from your first round of bee extractions, you can clean it up and concentrate it with a small spin column like the one in the Qiagen MinElute PCR purification kit. These silica-based columns will help get rid of inhibitors and allow you to elute your DNA in very small volumes. I would start with this and see how it goes.

5. The Quick-gDNA kit is made for mammalian cells. Your bees are high in mucopolysaccharides because of their exoskeletons. These compounds are serious PCR inhibitors and are often co-extracted with DNA. If you want to try a whole new round of extractions, look for a kit like a mollusk DNA extraction kit that specifically deals with this problem. E.N.Z.A. Mollusc DNA Isolation Kit (Omega Bio-Tek, Inc., Norcross, GA, USA) is a good one.

Good luck and feel free to follow up,

Meredith

Thanks guys