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DNA Extraction from buccal swabs


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#1 GradMom

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Posted 02 April 2014 - 01:26 PM

I have buccal swabs that were collected by a lab tech in the field. The species of concern is an ungulate, if that matters.

 

A labmate of mine had success storing identical swabs in TES buffer (100mM Tris, 100 mM EDTA, and 2% SDS), keeping them at ambient temperature, then freezing them at -20 when he got back to the lab before he extracted them. So, I instructed our tech to follow that procedure.

 

I've extracted a few of these swabs and so far I'm getting virtually no DNA whereas my labmate was getting 20-40 ng/ul per swab. I can't for the life of me figure out why. Perhaps you folks can look over my protocol and offer some pointers.

 

I'm following the protocol outlined on pg. 37 of THIS Qiagen pamphlet with the following changes:

As my swabs are already in 600 uL of buffer, I don't add PBS.

I'm increasing the amount of Protease K to 40 uL and the incubation time to 48 hours (per my labmate's advice)

I elute in 75 uL AE (per my labmate's advice)

 

I'm wondering if I need to add a salt to the TES buffered swabs initially but if so then why would the extraction have worked so well for my labmate? wacko.png

 

Should I abandon the kit and go for a phenol/chloroform extraction? Would it be at all useful to remove the swab from the TES to a tube with PBS and use the Qiagen protocol verbatum?



#2 merlav

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Posted 03 April 2014 - 07:56 AM

Wich one of the Qiagen kit you are using? I can't open the link, usually for the qiagen kits you must increase the time of centrifugation.  I used to extract DNA from buccal swabs and the important tips for a good extraction are: the person must not eat at least 15-30 min prior the sample take (specially coffee), must rinse several times and instruct the person that samples are from gums not teeth.  let the swab air dry and can be keep at RT.  At lab make sure that you rinse the swab into the buffer very well to loose the sample, with the border of tube try to squeeze back all the buffer that you can.  then you can freeze until use.  If you are using Proteinase K of qiagen maybe you are using too much also 48 h for a sample that isn't hard like tissue is too much time also  


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#3 GradMom

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Posted 03 April 2014 - 08:39 AM

I'm using a blood and tissue kit, which is what my labmate used. How much are you increasing the centrifugation time by?

 

These are buccal swabs from pronghorn antelope. The cheeks were swabbed and then the swab was ejected into a tube with buffer. Knowing what I know now, I would have preffered that the swabs had been allowed to dry. That might have also caused problems as the area where the swabs were being collected was quite dusty and chaotic. I've been adding the Proteinase K to the tube containing the swab and then letting that incubate for 48 hours. Would it be better to maybe vortex the tube with the swab & PBS really well to release any sample into the buffer then squeeze the swab out and remove it? I've been afraid to remove the swab before the digestion.



#4 merlav

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Posted 04 April 2014 - 04:38 AM

laugh.png I thought that you were sampling people.  Ok try to let it dry a min or 2  and cap, then at your lab keep at 4C until extraction (I recommend that you don't let it too much time maybe 1 or 2 days).  1st. use the buffer that comes with the kit and with your thumb and index gently roll the swab in the buffer to loose the cells about 20x  then squeeze (keep the swabs in pbs just in case you need to go back) .  2nd. add the proteinase K and mix well by pipetting or gentle vortex, 3rd incubate no more than 30 min at 55C (If my memory don't fail thats the temp for qiagen proteinase), checking ever 10 min and vortex to make sure that samples will digest.  I think that 48h is too much for cells it may be destroying too much.  I use such a harsh condition for tough tissues.  centrifuge for at least 1 min instead of the 30s that usually the qiagen kits use. At the final step add the buffer in 2 step instead of one (add half the volume to the column, incubate 3 min centrifuge, repeat).  Hope this help!! 


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#5 GradMom

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Posted 07 April 2014 - 08:30 AM

Thanks. My samples are already collected. I don't have the option to go back and collect them dry. Maybe next year but for now I've got about 100 swabs sitting in the freezer in TES buffer sad.png



#6 pito

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Posted 07 April 2014 - 11:48 AM

the problem could simply be that you did not swab well enough and that there are not enough cells in your samples....

 

 

Ask the labtech to do his protocol (extract the DNA) on some of your samples in parallel with his samples. This way you can see if its you doing something wrong with the protocol or that your samples are bad.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#7 merlav

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Posted 07 April 2014 - 03:12 PM

I'm unfamiliar with the TES buffer, you are using it as a storage buffer or a lysis buffer?
Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#8 GradMom

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Posted 10 April 2014 - 10:07 AM

I'm unfamiliar with the TES buffer, you are using it as a storage buffer or a lysis buffer?

 

Both. My PI insists on using this buffer. I would have prefered to collect the swabs dry and then just added PBS when we got back to the lab.






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