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Shrimp nuclease to eliminate genomic DNA - (Apr/07/2009 )

We are using RNA derived from sheep and cattle in RT-PCR but are finding evidence for genomic DNA contamination. We have tried various DNAse clean-up reagents but still find evidence of contamination. I have heard that shrimp nuclease may be a good way of getting rid of ds DNA. Has anyone used this and if so, what was the method that you used?

Thanks

willia

-willia-

I always use RNeasy plus mini kit from qiagen which has a gDNA eliminator column for RNA extraction and find no contaminating DNA, u can try using it for your RNA extraction.

-mtrnbh-

For RNA isolated with Qiagen kits we use Qiagen DNase on columns, and for those samples isolated from Trizol we use Ambion's Turbo DNA-free. We don't have any problems with DNA contaminations in real time.

-Trof-

Thanks for the replies but weve actually tried the Qiagen DNAse and the Qiagen clean-up method and we are still experiencing problems. Thats why we are attempting unconventional methods. Mainly its the sheep DNA thats the problem. Seems to me that the cell size is the main problem.

It seems that the shrimp nuclease may be a very good option... if it works. The patent claims good results and we will be trialing it next week. It seems that you add the nuclease to the RT step of a QPCR experiment and the shrimp nuclease has specific affinity for ds DNA. The reaction should be halted when the nuclease is exposed to temp above 65C. I really wanted to hear from people who have actually used the nuclease to see how it had worked in their hands since Ive not been able to find any record.

Thanks for your comments though.

-willia-

willia on Apr 7 2009, 09:33 PM said:

We are using RNA derived from sheep and cattle in RT-PCR but are finding evidence for genomic DNA contamination. We have tried various DNAse clean-up reagents but still find evidence of contamination. I have heard that shrimp nuclease may be a good way of getting rid of ds DNA. Has anyone used this and if so, what was the method that you used?

Thanks

willia


Try!
It's not so much used yet, simply because it has not been marketed very much.

You should use 0.1-1 unit. Works in most buffers (can be used directly in the RT-PCR together with primers and probes; Do not like SYBR!); needs magnesium, pH optimum is 7.5 (check your buffer), optimum temp is 40 C.
Recommended inactivation: 15 min at 65C in the presence of 1 mM DTT (100% irreversible inactivation). No need for removal of the enzyme.

It works very well in the systems where it has been tested, but has not (to my attention) been tested in sheep and cattle nucleic acids.
Can be bought from USB, Thermo Scientific, in some countries from GE Healthcare, or directly from the producer: Biotec Pharmacon (www.biotec.no)
Feel free to contact us for more detailed questions.

Gerd Nilsen (Product manager, Biotec Pharmacon)
Attached File

-Gerd-

Hi Gerd,
Thanks so much for the information. We are going to try it out this week.

I could not download your link. Can you attach it again?

Thanks

-willia-

Hi Gerd,

I tried to email you on your site at Biotec Pharmacon but the message would not send from your contact page. Please can you email details of the shrimp nuclease to the following email address a.purdie@usyd.edu.au.

We are really keen to try the shrimp nuclease and have purchased it from Thermo. Can you verify the experimental conditions for me. We are planning on adding it into the RT step as we do qPCR with SYBR. Do you think that the enzyme will still be inhibitory to the qPRC reaction even after the heating step in the RT process?

I could not download the PDF on your post, the file comes up as damaged. Please can you send it again?

Thanks

-willia-

Gerd on Apr 13 2009, 11:58 PM said:

willia on Apr 7 2009, 09:33 PM said:

We are using RNA derived from sheep and cattle in RT-PCR but are finding evidence for genomic DNA contamination. We have tried various DNAse clean-up reagents but still find evidence of contamination. I have heard that shrimp nuclease may be a good way of getting rid of ds DNA. Has anyone used this and if so, what was the method that you used?

Thanks

willia


Try!
It's not so much used yet, simply because it has not been marketed very much.

You should use 0.1-1 unit. Works in most buffers (can be used directly in the RT-PCR together with primers and probes; Do not like SYBR!); needs magnesium, pH optimum is 7.5 (check your buffer), optimum temp is 40 C.
Recommended inactivation: 15 min at 65C in the presence of 1 mM DTT (100% irreversible inactivation). No need for removal of the enzyme.

It works very well in the systems where it has been tested, but has not (to my attention) been tested in sheep and cattle nucleic acids.
Can be bought from USB, Thermo Scientific, in some countries from GE Healthcare, or directly from the producer: Biotec Pharmacon (www.biotec.no)
Feel free to contact us for more detailed questions.

Gerd Nilsen (Product manager, Biotec Pharmacon)


Seems like there is a problem downloading my attached PDF. If you want it; please send me a message ( gn@biotec.no) and I can send it directly to you.

-Gerd-