hygromycin and arabidopsis

Michael J. Prigge via methods%40net.bio.net (by mprigge from indiana.edu)
Wed Oct 31 23:34:47 EST 2007

I would guess that the false positive could be due to one of two  
reasons.  You could blast your primers against the Arabidopsis genome  
to see if, by chance, there is 500 bp region flanked by sequences  
similar to one or both of your primers.  You could also check the  
sequence or restriction pattern to confirm that the 500 bp fragment  
is what you expect.  More likely, one or more of your pipettors is  
contaminated (from pipetting the plasmid or PCR products).  Did you  
do a no-DNA control?  If you also see the product, then you'll  
probably have to replace your reagents and clean your pipettors  
(especially inside the shaft) or borrow someone else's for setting up  
the reactions.  (UV treatment has been shown to reduce contamination,  
too.)  Pipettor contamination can happen surprisingly easily,  
especially if you pipet up and down to mix in dye or to rinse tips  
between loading samples on the gel.  I routinely use a different  
pipettor for loading PCR products on gels.
Good Luck,

> SVemuri12 from gmail.com via methods%40net.bio.net (by SVemuri12  
> from gmail.com)
> Tue Oct 30 17:45:39 EST 2007
> I am working with wildtype and transformed lines of Arabidopsis and am
> using hygromycin as an antibiotic for selection. On my plates, the
> transformed line show good hygromycin resistance, growing into full
> green healthy plants with roots. The wildtype line shows good Hyg
> sensitivity. So, I knew my antibiotic concentration was working.
> Then, I ran the same lines on PCR using Hyg primers. I grew up WT line
> separately on soil since they would never survive the Hyg antibiotic.
> But I took the transformed lines striaght from Hyg plates and
> transferred the ones that survived the antibiotic right to soil. I
> prepared genomic DNA after the lines grew old enough.
> Now, the PCR shows products in both WT and transformed line. The
> product size is the same on both lines, about 500bp. I tested all my
> reagants & ran various gels with different annealing temps. But I get
> the same products every single time. Does anyone know what this could
> mean to see PCR product on the WT line when this line has previously
> not shown to be resistant to hygromycin on the Hyg plates? is this
> supposed to happen?
> Thanks.

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