Having previously and successfully used the In-Fusion cloning kit I'm having real issues with my current round of cloning. I have a number if 2kb-ish fragments that I'm trying to insert into the EcoR1 site of the pEF-IRES bicistronic vector. The inserts amplify without issue, the vector linearises without issue, I even see a band of the correct size after the 'ligation' BUT.......no colonies! This is using both the Stellar and and TOP10 cells. Controls work fine. All primers designed in direct accordance with the instructions. I've checked the sequence of my plasmid prep. I've tried gel purification verse Cloning enhancer for the amplified fragments (which they say ges rid of template.....which it doesn't......and I don't see how it would....as an aside, can anyone give me the heads up on this, have I missed something very basic?) but to no avail. So........anyone had any similar issues who could pass on their wisdom?
Desperate enough now to consider any ideas!!
Hi, I am using In-Fusion system and I also would suggest that you design longer primers. Usually for design, I am using Snapgene program (you can download 1 month free trial). Otherwise you can also use a similar kit from NEB. Just to add , for me In Fusion with cloning enhancer works better compared to gel purified DNA fragment. Good luck!
This might not help you now, but might help a lot of other people. Your insert might be getting looped out because of instability. Try using a different strain of E.coli like Stbl2 or Stbl3 or NEB Stable or XL10Gold. I had a similar issue and was doubting the inFusion reaction but it turned out to be an issue with the stability of my gene.
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