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genomic DNA isolation


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#1 umam

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Posted 04 March 2010 - 06:15 AM

Hi,

I am trying to isolate gDNA from mosquito larvae that have been fed with bacteria. I was using the Qiagen blood and easy kit and it suddenly stopped working. I can't even isolate the mosquito gDNA (verified by actin). I tried to use the Zymo kit but did not see any change. I did not make any changes in the protocol, so I am completely clueless as to why it stopped working. I tried using a different Qiagen kit (same kit from another lab to make sure that all my reagents were working fine). My PCR seems to works as I always see my positive control (plasmid DNA).
I tried increasing the amounts of lysis buffer and proteinase K but still did not see any results. I am stuck from the past couple of weeks, please post any suggestions that you have. Also, I preferably do not wish to use phenol:chloroform method. I need to use the gDNA for real time PCR.

Thanks,
-Uma

#2 pDNA

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Posted 04 March 2010 - 12:05 PM

Hi,

I am trying to isolate gDNA from mosquito larvae that have been fed with bacteria. I was using the Qiagen blood and easy kit and it suddenly stopped working. I can't even isolate the mosquito gDNA (verified by actin). I tried to use the Zymo kit but did not see any change. I did not make any changes in the protocol, so I am completely clueless as to why it stopped working. I tried using a different Qiagen kit (same kit from another lab to make sure that all my reagents were working fine). My PCR seems to works as I always see my positive control (plasmid DNA).
I tried increasing the amounts of lysis buffer and proteinase K but still did not see any results. I am stuck from the past couple of weeks, please post any suggestions that you have. Also, I preferably do not wish to use phenol:chloroform method. I need to use the gDNA for real time PCR.

Thanks,
-Uma


U are using 2 different kits, from 2 different manufactures ...to my point of view something else than the kits seems to be the cause for your problems!

Try to purify some standard E. coli culture as a kind of positive control ...so you can be sure that the kits are working fine.

Maybe it is due to the media u use for cultivation of your larvae? maybe something has been changed? (pH, salt concentrations, etc)

Regards,
p

#3 umam

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Posted 05 March 2010 - 06:44 AM

Yes, that's what I think, I am positive that it's not the kits, but at the same time,
the pH, salt etc has not really changed and I get good gDNA isolation from bacteria using the same kits.
-uma

Hi,

I am trying to isolate gDNA from mosquito larvae that have been fed with bacteria. I was using the Qiagen blood and easy kit and it suddenly stopped working. I can't even isolate the mosquito gDNA (verified by actin). I tried to use the Zymo kit but did not see any change. I did not make any changes in the protocol, so I am completely clueless as to why it stopped working. I tried using a different Qiagen kit (same kit from another lab to make sure that all my reagents were working fine). My PCR seems to works as I always see my positive control (plasmid DNA).
I tried increasing the amounts of lysis buffer and proteinase K but still did not see any results. I am stuck from the past couple of weeks, please post any suggestions that you have. Also, I preferably do not wish to use phenol:chloroform method. I need to use the gDNA for real time PCR.

Thanks,
-Uma


U are using 2 different kits, from 2 different manufactures ...to my point of view something else than the kits seems to be the cause for your problems!

Try to purify some standard E. coli culture as a kind of positive control ...so you can be sure that the kits are working fine.

Maybe it is due to the media u use for cultivation of your larvae? maybe something has been changed? (pH, salt concentrations, etc)

Regards,
p



#4 Denny

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Posted 05 March 2010 - 07:07 PM

I just hate when this happens! Sometimes we never figure out what the heck went wrong and things just start working again.

Have you gone back and started fresh cultures of larva with fresh bacterial cultures?
Double check your media components, see if you've gotten a new batch, maybe different brand.
If the kits are working fine and PCR is working fine it seems to point to a change in the cultures.

If you can't find a solution save time, start fresh with all new instead of trying to rule out everything. I will go through my bench and clean my pipettes with "DNA Away" by ISC bioExpress to get rid of any DNA and DNASE contamination as well as replace the stock solutions at my bench.

Good Luck
We've all been there :D

#5 umam

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Posted 06 March 2010 - 07:22 AM

Exactly, I am going to try and start fresh as of nothing was wrong and repeat everything. Thanks for the DNase information. Will try that out.
Will keep you all posted, keep posting any suggestions you have.
Thanks,
-Uma

I just hate when this happens! Sometimes we never figure out what the heck went wrong and things just start working again.

Have you gone back and started fresh cultures of larva with fresh bacterial cultures?
Double check your media components, see if you've gotten a new batch, maybe different brand.
If the kits are working fine and PCR is working fine it seems to point to a change in the cultures.

If you can't find a solution save time, start fresh with all new instead of trying to rule out everything. I will go through my bench and clean my pipettes with "DNA Away" by ISC bioExpress to get rid of any DNA and DNASE contamination as well as replace the stock solutions at my bench.

Good Luck
We've all been there :lol:






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