What exactly are you trying to amplify, and what do you mean by 'results are not completely good'?

First of all, are your primers and reagents ok? Do you have template DNA that you can use to test if your PCR reagents are ok? If you are sure that your primers and other reagents are ok but you are getting variable results using the same PCR master mix on your actual samples, it's possible that your extracted DNA contains some compounds that interfere with the reaction - either from the coral or the extraction method. There are various methods to get cleaner template. Or, if you are unlucky, your template is degraded and you can't really do anything about it.

Hello Sebastian,

I had worked with the corals DNA and have experience same like your case. Sometimes, the DNA extraction for corals is tricky. In my work, I use Qiagen for DNA extraction but as I noticed before, it's tricky. Like CTAB method, maybe you should modify the amplification protocols to gain the "good" amount of DNA. The another problem that may be occur is come from your sample. Sometimes, there are contaminants which makes your extraction or amplification not done well, just like Paul said or try to recheck all your reagent used for extraction and PCR. If your reagents are still in excellent condition, try to extract the DNA again and if that still fail or try another extraction method, maybe kit or try to make some optimacy in your PCR protocol.

Hi Sebastian,

I agree with Stevanus and Paul.

Just to expand somewhat:

I work on soft corals conducting DNA isolation and amplification protocols. I have used CTAB extraction and got substandard template. The Qiagen blood and tissue kit is good as is the inexpensive Zymogen gDNA animal tissue kit (also the MN Nucleospin kit). Your tissue storage of said samples as well as homogenisation should be your initial trouble shooting focus. I find that the type of tissue storage (ethanol at room temp. vs -20 degree celsius vs -80 degree celsius) certainly affects the success of your isolation (in that order). Furthermore, the type of tissue used (polyparium is polyploidy) is important as well as age of tissue: obviously the fresher the better.

If you manage to obtain a kit for DNA isolation then ensure you perform all the 'extra' steps recommended for difficult organisms i.e. if it says to repeat a step just 'in case' then do so. If the digestion step takes takes 1-3 hours and there's an 'allowance' to leave it overnight then rather do so. These instructions are most likely in smaller print or on a different page depending on the manufacturer so read carefully. Remember to homogenise your samples thoroughly before extraction, depending on your resources you could use liquid nitrogen with a pestle and mortar or an actual homogenizer.

Amplification is another troubleshooting process altogether. It is impossible to optimise this process until you are certain that your template is of decent quality thereby saving yourself time, money and sleepless nights. It is a hack to re-isolate everything so take 5 samples and see if you cant borrow a kit from someone to try determine wether it is the template quality that is affecting your PCR. Unfortunately certain corals contain polymerase inhibitors that further complicate amplification.

My advice would be to focus on obtaining quality template before attempting further amplification.

Everything of the best to you!

Hello Dear Kerry,

Thank you for your advices. Actually they are very useful. The last weeks we were performing both the CTAB and the QIAGEN blood tissue minikit. So far the QIAGEN yielded better results. The PCR also was succesful, but we want to optimize it to get more clear bands. I am a bit curious about your work with soft corals, since I will work with indopacific scleratinian coral under lab conditions. Briefly, my PhD work wants to determine how corals will cope with predicted future ocean conditions, particularly pH decrease and temperature increase. The project is called "Ocean 2100" which is somehow a challenge for our group, since it will be our first approach to the genetic responses and for me as well, because during my master in the Colombian Caribbean my focus was on the ecological factors.

Best regards from Giessen, Germany.

Sebastián Celis

Dear Sebastian

Your work sounds very interesting! There are so many of us working on different aspects of the genetics of corals I feel sometimes that a support group is in order. From my own experience I found that crossing over into the molecular world was not actually as intimidating as it seems.

Paul's pointers are spot on. You haven't mentioned what you are trying to amplify or the protocol used- i.e. are your primers novel or based on previous literature? Have they been used on these species previously?

When you say that you're amplicons are 'ok' what do you mean by that- are you getting primer-dimer, multiple bands or smears? Do your controls work? What steps have you taken towards optimisation thus far?

There are a lot of variables to consider therefore the more information provided the more useful the suggestions. Please don't hesitate to email me either.

Warm regards

Kerry

Dear Keery-Lee,

Thank you for your comments, Right now I am solving the puzzle of my PhD project. As I already mentioned, I will be focusing in genetic stress mechanisms under predicted pH and temperature values. The literature reports diferential gene expression for several scleractinian species, both under lab and field conditions. We will be working with an experimental setup which will simulate those conditions and also the natural fluctuations due to p.e. up-welling.

I am wondering whether in your group have worked with either octocorals or scleractionia under laboratory conditions, since I want to gain information regarding other experimental setups and also about relevant information gaps to be fill up about coral stress response. I am thinking that cDNA microarrays would be a good tool in order to elucidate the particular response for our corals, specifically Porites rus, whcih will be our "laboratory rat"

This is my email address, if you want to share relevant and useful information. As you can see, I am looking for feedback and insights from other groups like yours. [email protected]

Saludos desde Alemania. Gruesse aud Deutchsland,

Sebastián

Hi Sebastian,

We recently did a blog post about how to extract DNA from coral that might be helpful to anyone on this thread :)

https://www.zymoresearch.com/blogs/blog/can-coral-dna-measure-ocean-health

Thank you Sarah!