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How much transgenes should appear?

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#1 Mufitdhahnurul

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Posted 01 May 2018 - 08:48 PM

Hallo to everyone.

 

I have tried to do southern blotting to see transgenes on overexpression plants. I am using DIG high prime kit II from Roche and cutting dna using HinDIII endonuclease. The probe is made by the pcr method using a primer that can recognize the target gene. in this case, I assume that this plant has 2 endogenous and exogenous genes of the genetic transformation process. Southern blot using a probe with primer gene resistance to antibiotics (probe a) has also been performed. the result looks one fragment in the transgenic plant, but no bands appear on the wt. After that, a southern blot using southern blot using a primer that can recognize the gene target  (probe B) also shows only one fragment dna in all samples including wt.
1. whether the fargment that appears can be said as a copy number?
2. should not two fargments appear with probe b, since the sample contains both endogenous and exogenous DNA?

please give me explanations and suggestions for my problem this-
terimaksih
-Mufit-



#2 bob1

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Posted 04 May 2018 - 06:42 AM

For each gene that you are trying to detect, are the fragments different sizes? If so, how much difference is there - I would expect that the genes would run at the same size so you would have to look for a band that is twice as bright.

 

It could also be that your washing conditions or probe binding conditions are too stringent. - try annealing at a lower temperature and washing with less salt.







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