Our lab is facing problem in recombining >4Kb insert with 6Kb vector.
We are using 30-40 base pairs overhang in our insert to facilitate recombination.
We tried Gibson assembly mix from NEB, Metamorph mutagenesis mix from SBI and Infusion mix from Clontech.
The basic observations, after we sequenced the transformed plasmids, are that the initial and end regions of the insert gets ligated/recombined with overhang sequences at correct position in the vector but the huge middle chunk gets missing. So we basically gets a truncated version of insert in the vector and not complete insert.
We only face this problem when we use big size inserts.
Kindly help me with your experiences and suggestions.
Hi, you should check that your insert does not contain any repetitive sequences. If so, the bacteria is able to cut out the regions containing that repetitions. It's common problem in cloning lentiviral vectors containing LTR (long terminal repeats). To prevent the problem you should use recombination deficient cells like Stbl3 from Thermo/Invitrogen https://www.thermofisher.com/order/catalog/product/C737303 . The cultivation of bacteria at 30C also decrease the risk of recombination and deletion of your insert. Good luck,
petr
Hi, you should check that your insert does not contain any repetitive sequences. If so, the bacteria is able to cut out the regions containing that repetitions. It's common problem in cloning lentiviral vectors containing LTR (long terminal repeats). To prevent the problem you should use recombination deficient cells like Stbl3 from Thermo/Invitrogen https://www.thermofisher.com/order/catalog/product/C737303 . The cultivation of bacteria at 30C also decrease the risk of recombination and deletion of your insert. Good luck,
petr
Hi Peter, Thank you for your look out. We tried transformation using STBL cells from thermo but the results were the same. Meantime we tried fragmenting the long single insert and then tried recombining them using Gibson and Metamorph mutagenesis mix and Voila! we achieved cloning the >4kb insert in 6kb vector.
I believe in your words that there could be repetitive regions which gets cut out during recombination but when I checked manually, there was no such long repeats that could behave like LTRs. Though we completed our experiment but still wants to know if there are some kits or modified protocol which helps in intyegrating bigger inserts in the vector.
Hi Shruti Chitransh,
I totally agree with Petr Muller as I had the same problem then I solve it by using Stbl3 cells. In your case now, how did you get your insert? have you checked the sequence before the cloning? If yes, then I think you may try the Hi Fi assembly.
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