I've had great success extracting DNA from ticks (which is way harder to perform than with beetles) using Qiagen blood and tissue DNA extraction kit. It's about $200 for 50 samples. 

So I was going to give you the same feed back as Michael. The Qiagen blood/tissue kit works well for tick DNA extraction. Tylpically to break apart the ticks I would use larger glass beads and a ultra centrifuge. Down streak applications (qPC and Sequencing) worked great with it.

Dear Heather and Michael, I'm looking for a cheaper way to do it. The Qiagen kit is really expensive for me, I can't afford it.

Anyway, thank you very much.

I finally extracted DNA from adult beetles using a CTAB protocol. In this protocol, I only used chloroform and omitted phenol. But I think there is still a problem, something is stuck in the well. I will start a new topic about it and share the link here.

I had success with tiny ambrosia beetles using PrepMan Ultra by Thermo Fisher. It's a single-step, single reagent extraction kit - just aliquot the buffer, add material and heat, and you're done. It's also really affordable.

I pinched off a single leg, crushed it a little bit with a needle, and extracted using a thermocycler (100C for ten minutes is all it takes). Doing this in 50 microliters of PrepMan was good enough to amplify COI with just one microliter of template without concentrating. I can't verify whether or not it works with single-copy genes or in other insects.

It shouldn't really matter which protocol you are using. I have used Qiagen (presumably the best), EZNA - a cheap Chinese brand, and salting out which is nearly for free, and everything work well on bark beetles. The beetles you mention are big. Perhaps you are using too big chuncks of tissue which make the extract dirty and probably full of PCR inhibitors. I usually have trouble with larger beetles above 3-4 mm long. The smaller, the better. You only need 1 sq. mm of tissue, or even less. One other problem with big beetles is that they are often not well preserved because the ethanol does not reach very far into the body. Therefore it is advisable to take tissues where the body is broken. If you take legs and no muscle tissue is visible, I would crunch them to make access to the tissue.

Hi Professor Jordal

Thank you very much for your answer.

That's right. Yes I used the legs for extraction (about 1 to 1.5 cm)!

I trusted these two videos:

https://www.dnalc.org/view/16959-DNA-Barcoding-Protocol-Isolating-DNA.html

https://www.youtube.com/watch?v=bMgkMroXD5U

They say, we can use a hind leg of a grasshopper for DNA extraction, which is obviously big.

I store the specimens in the fridge (at minus 20). So, tomorrow I will try just a small piece of the leg.

I created a new topic about my DNA contamination, you can follow it here:

https://www.researchgate.net/post/Pigment_contamination_in_DNA

Hi again

Dear Professor Jordal, I tested just a small piece of the leg, but I didn't see anything on the agarose gel (1%). I cut a small piece about 1 mm (femur) and then put it in the microcentrifuge tube and added the CTAB buffer and crushed the tissue using a pestle (of course it was really hard to crush).

But I extracted DNA from an Amara aenea (about 7 mm), the DNA is great, but I'm afraid of cross contamination of the gut content, of course I captured the beetles still they were alive and then after 2 or 3 days killed them in the fridge at minus 20, so probably the fear about cross contamination is not really serious, of course sometimes they were together in the same container :-(

Any suggestions?

So your 1mm of the femur was cuticle? or inner muscle tissue? The point is that well preserved muscle tissue must be used. If so there should be no problem. I never run DNA on a gel, just doing the PCR.

Thanks Professor Jordal

I simply cut a piece of the femur and crushed it in the CTAB buffer, I don't know how I can use the inner tissue separately!

Is there a standard procedure to remove the cuticle to achieve the pure muscle tissue?

I did the protocol accurately, and I also used a very fresh tissue (I got the insect about 1 hour before extraction). But I didn't see anything on the gel, of course I know that sometimes it doesn't matter! because agarose gel is not very accurate and running an acrylamide gel should be better or directly doing the PCR.

You don't need pure muscle tissue. But you explained that you used 1 mm of leg, which sounds like mainly cuticle because of the size of the insect. My point is that you must be sure that i) there is muscle tissue available, and ii) the tissue must have been dehydrated properly by ethanol when killed (not applicable if frozen alive)

Based on my morphology knowledge, femur is rich of muscles, especially in ground beetles. I think the tissue is available, but I didn't save the beetles in ethanol, I directly killed them in the fridge. I think there might be a way to dehydrate them again.

On the other hand, I have DNA from Amara aenea (whole)and Myriochila melancholica (just thorax) which were frozen 3 months ago!

I will search about the techniques which used to dehydrate frozen tissues, and I'll share the results here.

Hi again

Yesterday I did the PCR and I had good DNA bands. I'm still afraid of DNA contamination, I think I should find a way to extract DNA from legs.

You can see the gels that I attached. Excuse me for the low quality images, I took the pictures from the CRT-Screen using my mobile (cellphone), for some reasons I don't have access to the main pictures that I saved on the gel doc computer.