I've been using Golden gate method to create plasmids. I've mini preped and sequenced a plasmid and it was confirmed to be correct. I took this mini prep DNA transformed it and used it to do a maxi prep and my maxi preps are for some reason not assembling correctly. It's so frustrating because when I mini prep this DNA it's fine but I've never gotten an accurate maxi prep from it. This isn't the first plasmid I've had this issue with. I just don't understand how using the same DNA that has been confirmed to be correct for both maxi and mini the assembly isn't working for maxi.
Dear Marie Skinner ,
May be there is a miss understanding from my site.
So your problem is that you have done a GGA cloning and your Mini-Prep is correct and was confirmed by sequencing but you are not able to make a Maxi Prep from it? Do I understand you correct that you are transfroming the Maxi Prep bacteria again(!)? Why did you then do the sequencing, since chances are high that anything else is than in your bacteria.
The work flow should be (in my opinion):
- Transformation
- single clone agar plate with selection Amp/Kana/what ever is coded in your backbone
- Pick some clones for mini preps (i.e.)
- over night culture (I use 6 ml)
- miniprep of 4 ml followed by restriction analysis (to estimate the size or identify your insert by a third restriction site) keep the rest 2 ml in the fridge
- sent out 1-3 samples out for sequencing
- get the correct results from i.e. klon 2 within some days or 2 weeks
- use than 200-500 µl of the clone in the fridge (do not store them longer than 4 weeks) to start your Maxi-Prep and culture over night
- do the maxi-prep on the next morning
Have I understand you correct? Is the second transformation (for the maxi-prep) your problem?
Best wishes Soenke
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