Hi,
I collected buccal swabs (cotton and foam) from nestlings and adult birds and stored the swabs in 70% ethanol. There are few different protocols on the web but I am wondering if anyone has a detailed protocol that they would be willing to share with me.
I am a bit confused when to remove the swab and if I treat the buccal tissue as tissue and add the ATL buffer from the kit. Some protocols do not use the buffer ATL for swabs while some papers say they followed the manufacturer's manual.
Thank you!
Hi Jessica Gutierrez
I hope the answer is your question.
Article Evaluation of the impact of six different DNA extraction met...
http://biotools.eu/documentospdf/Speedtools%20Tissue%20DNA%20Extraction%20Kit.%20eng.%20Ed%2008.%20February%2016.pdf
Read page 19.
First crucial step is to let the ethanol evaporate completly (otherwise it will block the proteinase in lysis step). Ideally if you have a flow box and single use petri dishes - each swab to be placed on one petri dish. Manipulate the swabs with tweezers, after each swab I would reccomned to burn the tip of the tweezers in flame (avoid cross-contamination), let cool down and proceeed to next swab. When the swabs are fully dried out, you can proceed to isolation of DNA. All lysation steps are done with the swab of course, addition of buffers for tissue lysis does not any harm. But before the step "addition of 96-100% ethanol" I would greatly recommend to move all liqid to a fresh, empty 1,5ml tube. Ethanol causes coagulation of the DNA (and likely other stuff), the DNA could get stuck in the cotton and you would loose it. When the sample liquid is in the new tube, add the ethanol and proceed according the protocol. Make sure you preheat the elution buffer at the end, it greatly improved DNA gain.
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