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PCR failing for transformed plant DNA but not for Agro

PCR Transformation Arabidopsis gDNA Agrobacterium

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#1 pizzahutt

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Posted 17 September 2013 - 11:14 AM

Good Afternoon,

 

I have been struggling with PCR for a gene that a former coworker had transformed into Arabidopsis.

 

About 15 months ago, I was given a bag of Arabidopsis seeds that had been transformed previously by a former lab member. I had isolated what I believed to be a homozygous line for the gene of interest through planting several generations of the plants on herbicide media.

 

Upon testing for presence of the gene of interest with PCR, I have been unable to get a positive result ever. I have tried adjusting the annealing temp, using different polymerases and kits, and different methods of extracting the genomic DNA. I have used the agrobacterium that was used to transform the plants as a positive control and can almost always get a band.

 

I understand that the agro has such a high concentration of the GOI and that may be why I am able to get a band but not for the gDNA.

 

Could someone help me with this issue? Most of the folks in my building are pretty busy with their own projects and don't have time to help me troubleshoot this process. Any insight would be appreciated.

 

Thank you.



#2 phage434

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Posted 17 September 2013 - 06:22 PM

How are you isolating DNA from your plants? Can you amplify other genes from your isolated DNA?



#3 pizzahutt

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Posted 17 September 2013 - 07:30 PM

How are you isolating DNA from your plants? Can you amplify other genes from your isolated DNA?

Thanks for responding. I have been able to amplify other genes that were transformed into arabidopsis by my former lab member at the same time with relative ease, I am struggling with only one gene. 

 

I have tried using the Qiagen miniprep kit, as well as different leaf or seedling preps with different variations of Edward's buffer. The Qiagen kit gave me a good amount of DNA with a A260/280 purity of around 1.6 but I still saw nothing, even after adding more or less template DNA. But I always got a band for the agro sample.



#4 phage434

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Posted 18 September 2013 - 01:46 PM

A miniprep of plant tissue will (at best) yield genomic DNA as a contaminant, not as a product. I'm not familiar with Edward's  buffer, but I would suggest a CTAB extraction followed by phenol/chloroform extraction and cleanup with chloroform only and ethanol precipitation. This is a very standard plant genomic DNA prep.







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