I am trying to make a plasmid by traditional cloning. The size of the insert is 7.3 kb and the size of the vector is about 7.2 kb. I am using PacI to isolate insert and prepare the vector. I had a successful ligation but when I transformed it to the E. coli strains that we have (Top10, NEB stable beta competent cell, Bioline alpha competent gold efficiency). It seemed that the plasmid is recombined and rearranged. Is there any specialized cell lines which I can use for this difficult cloning. Also if you have any suggestion regarding this cloning please let me know.
Have you sequenced the plasmid? You should check if there are any integration sequences. If you have some you should use a strain defected in recombination systems.
check the sequencing first as your insert and vector are about the same size and then once you have confirmed that your sequence is inserted make sure that it is in frame. Try using NEBs T7 express as you will be able to prepare plasmid and over express protein in the same strain.
Please checked your vector/inserted seq junction with a simple pcr for two aim 1_insertion 2_correct direction of your insert...
If you have control vector..you can checked your cloning process
Note: restriction enzyme in 5&3 prime end of insert/ligate can show your direction.
For proprate bacteria strain...top10_DH5 alpha_jm109...can be responsible
Occurrence of recombination of a DNA construct has been reported. However, your competent cells, Top10, NEB stable beta competent cell, are specific to be used for reducing 'recombination' (ex. between repeats of a construct). They have a recA1 genotype to reduced recombination of cloned DNA.
https://www.neb.com/products/c3040-neb-stable-competent-e-coli-high-efficiency
I am wondering: how do you know that your construct has been rearranged or recombined during to recombination?
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