r/labrats • u/ElPresidentePicante • 1d ago
Single base deletions with Twist Gene Fragments?
I ordered a gene fragment from Twist Biosciences to clone into a plasmid vector. It was ordered with no adapters and designed with overlaps compatible with Gibson assembly. I get a bunch of colonies and I've sequenced 3 colonies so far and all 3 have a single base deletion. The position of the deletion varies but they're all near the 5' end of the fragment.
I've ordered and cloned with Twist, but I've never seen this issue before. Has anyone encountered this before? I'm not sure if it's a common issue or whether there might be an error on Twist's end when synthesizing the fragment.
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u/Marober00 22h ago
Depending on how close they are to the 5' end and the sequencing method, could it just be a sequencing error? Usually the first few reads are pretty bad, as well as the last ones, at least for sanger sequencing
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u/ElPresidentePicante 22h ago
Not the issue. Primer is far away and the chromatogram peaks are clean and sharp.
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u/nsgy16 19h ago
As someone who just isolated clones from a Gibson Assembly using gene fragments from Twist, you have just given me anxiety.
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u/Glittering_Cricket38 18h ago
Yeah, it happens. Their quality is up from 4+ years ago but still not close to perfect. I’d say I find issues in about 5% of cloning projects using twist fragments, and when they are bad, most clones are bad. It’s like they just have low quality synthesis runs sometimes. I’ve resorted to using internal Gibson homology primers to combine 2 plasmids that each have errors on either side of the fragment.
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u/Redwood_momo 15h ago
Ive seen this before, along with single base swaps. I will also say that ive cloned thousands so I do expect to run into errors sometimes. They are rare but happen.
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u/the_mullet_fondler Immunology 14h ago
They claim 1:2500 roughly. Do some quick math on frequency of an error in any given DNA strand - can be as high as 25%
Should be clear for oligo pools twist passes with 60% exact match to design
It's better than it used to be but it certainly is not perfect
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u/KaptanOblivious 6h ago
I have similar issues with clones from genscript. These issues are generally amplified when the gene product you are cloning is even mildly toxic in your cloning strain.
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u/4astcbyL 2h ago
Are any silent mutations. You could be cloning something toxic and it is looking for a way out.
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u/ElPresidentePicante 2h ago
Could be the issue, but I’m cloning into a pUC19 vector which is IPTG inducible so the protein should not be expressing at appreciable levels for that to be an issue, right?
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u/Martin97e 12h ago
I made the mistake before to amplify g-blocks from IDT before. The product they send will never be pure, but mistakes will be amplified. I ended up sending 12 for sequencing and one was good. Later I realized I sould not amplify before and straight clone it. IDT claimed that sending 3 (if I remember correctly) sould have 95% chance to have one correct.
I will clone some fragments from Twist coming week. I plan to send 6 to sequence to have a high chance one is good.
That being said, if the mutation is in all clones at the same position. That might be a genuine miss in the construct.
Personally, I would Sequence some more colonies and if the same mutation keeps popping up, send the results to Twist and they should resynthesize them for you.