Hi all, working on some HIFI assemblies and wondering if anyone could give me some tips. I am struggling with getting an insert of about 2.5 kbp to ligate to my vector of interest. I have done several other inserts with success but this one is giving me a lot of problems. I have an overlap region around 23 bps (annealing temp 68 for Q5) on either end of the insert that I have been able to use regularly for other inserts (these inserts are all going into the same plasmid individually). I am able to amplify the gene using Q5 and it is very clean so I do not gel purify as I always seem to lose all my DNA in the purification process. All the other pieces I have put in this plasmid are shorter (1.5 or less kbp) and seem to work most of the time and are really forgiving. I leave my HIFI for a long time (4 hours) because I found that I get a lot of off target colonies if I don't. I do HIFI in a variety of ways, sometimes it works straight from Twist's synthesized genes I get without even amplifying them, other times it requires me to purify out the PCR and quantify it, but I always normally get a successful colony. I am really stumped at this point as to why this insert does not want to go in the plasmid.
If you can design overlaps with an annealing temperature closer to 48C (15-20 nucleotides) you'll have better efficiency, but that's unlikely to be the problem since the overlaps work with other inserts.
Do you clean up your PCRs? Primers from the PCR reaction can cause problems in the assembly, if you have very long primers then they might not be completely removed by some column clean up kits.
Is it possible the assembly is working but the plasmid doesn't grow well in the bacteria? E.g if your insert is toxic to the bacteria, or if there are repetitive elements in the plasmid. You can test this by cleaning up the HiFi assembly and then attempting to use it as template in a PCR with one primer binding to the insert and one primer binding to the vector, then check the amplicon size on a gel.
Ciaran Daly thanks for the suggestions. I do clean up my PCRs, but I have found that many of my inserts I have not had to. I typically will run a HIFI immediately after my PCR to save some time and get one started, while then also purifying the rest in case that doesn't work. I have also been able to take an insert straight from Twist without amplification and HIFI assemble it with these same overlapping ends. There is no rhyme or reason as to when it necessary to purify vs when I can just mix things together without purification. perhaps the annealing temp is the majority of my issues. Thanks!
Catherine Anne Kemme yeah I agree, it's not clear to me either why some Gibsons work with no clean ups, basically just throwing components together in a tube, whereas others require careful clean ups and careful ratios of fragments in the reaction. I usually do my Gibsons a quick and dirty way, and then if it doesn't work I go back and redo it carefully.
In my hands when it does not work it is because I got something wrong in the sequences (plasmid or primers..) Did you test your construct on the NeBuilder software? it will indicate if your construct is possible.
PS: once also it was because of the wrong antibiotic!
I am using neb hifi for assembling puc19 vector with two inserts. I used vector concn as 0.04 picomole and both inserts of 470 and 480 bp with 0.08 picomole concn each and then added 2x hifi master mix. 10 ul and kept for 1hr incubation at 50 degree but after transformation I am getting no colonies, I am not being able to understand why
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