I am going to insert a 6.3kb fragment into a 5.3kb plasmid. I use SAM1 digestion to cut the blunt end on the plasmid which is CCCGGG. I designed a set of primers to amplify the target fragment which the primers contain 30bp for overlap sequence for ligation (using isothermal assembly). Apart from samples, I also did some control (transformation: intact plasmid / only target fragment / only SMA1 digested plasmid / isothermal assembly treated target fragment/ isothermal assembly treated and SMA1 digested plasmid). There is no colony for isothermal assembly treated target fragment and a few colonies for only SMA1 digested plasmid and isothermal assembly treated and SMA1 digested plasmid. On the other hand, there are more than three times colonies for isothermal assembly treated samples (SMA1 digested plasmid plus target fragment in 1:3 or 1:7 ratio). I randomly selected 20 colonies for colony cracking, however, none of them have a target fragment inserted. I am confused why there are much more colonies than the control but no fragment inserted. I am genuinely asking you for help, thank you very much.
Hi the insert is pretty big. You would expect a lower frequency. Blunt ligation is probably no ot help either as it easier to get intermolecular ligation than intermolecular ligation.You may wish to subclone in parts. I'm not sure about your isothermal, but I regularly use DNAworks to make primers with directional restriction sites and I try to use the HiFi NEB restriction enzymes. You could also try a "quickchange" procedurebecause you have such a large insert. Good luck!!
Matthew Edward Thornton Thank you for your sharing, unfortunately, there are a lot of restriction enzyme sites over my target fragment so I cannot use two restriction sites to do my ligation. I also tried "quickchange" aka site-directed mutagenesis to ligate the target fragment and the vector but there is no positive result so far. Thank you very much for your sharing again
Hello! Did you look at the isoschizomers in the appendix of the NEB catalog? You could PCR purify and add additional restriction sites with DNAworks. It's such a big insert the sequence may be off. Every time I've had a problem it's been bunk sequence. I try to use serial cloner (dnastar lasergene) if your institute has a license. If it's cool, you'll get it!! Sorry about the bad spelling in earlier post. I can't edit RG on the phone.
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