I want to clone 3-6 repeats of a 100bp annealed oligo (AO) into a vector. After hybridization of the AO I see distinct bands of 100, 200, 300, 400, 500, 600 and 700 bp on a agarose gel. I cut these band and eluate in water. Then I prepare a normal Ligation with a BsaI digested and zipped vector and the bands of 300 and 600bp. The result is that I found clones with 100 and 200 bp inside but never with 300 and 600 bp. Why? Are the repeats not stable?
Best regards
Markus
Does this happen after you transform the plasmid into E. coli and do a mini-prep ?
Hi Elena, I have designed a 4 base overhang for the BsaI site on every site of the 100bp long annealed Oligos.
The Protokol for formation of the concatemers ist very simple.
10 ul AO, 1ul Ligase, 2 ul Ligase Buffer, 1 ul dNTPs , 6 ul water
30 min on ice, 10 min at 70°C for deactivation
Loading 20 ul on an agarose gel.
That´s it.
Then I cut the bands and do a ligation with the appropiate vector.
It seems that the bands disapear during the elution out of the gel. If I do an PCR with the ligation as template then I have only a 100 bp band.
But why?
Markus Langhans you haven't answered my question yet but if this problem happens after you transform the plasmid into E. coli and extract the plasmid then there is a high chance that this happens because of the E. coli strain you're using. We had this in the past when we were trying to clone 6x DNA repeats, everything was fine until we extract the plasmid from E. coli where we ended up with 2x repeats only and sometimes only 1. Then we realized that it is from the E .coli strain where there is a mechanism that allow E. coli cells to remove repeated DNA sequence. In order to get intact DNA repeats you either have to use a special E. coli strain with reduced genome or slow down the metabolism of the E. coli strain you're using by growing cells at lower temperature (28C).
Best of luck
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