I am Trying to use Infusion cloning to prepare a mutant construct. I have been told by a student that used it before that I need 20ng/ul of linearised plasmid to proceed with the following reaction:
3ul linearised plasmid
1ul Mutant gene (PCR product) with infusion designed primers attached
1ul Infusion Enzyme
Incubate for 15 mins at 50C
I have tried several restriction digests followed by extraction using a
ISOLATE II Genomic DNA Kit - Bioline. And always seem to get a low yield of linearised plasmid the best I got yesterday was 10.2ng/ul which is half as much as recommended. I tried the Infusion reaction anyway and it did not work. I am thinking because the concentration of Linearised plasmid was too low. I have tried the restriction and extraction several time and always get 10-13ng/ul.
I feel like I am burning through both time and kit, any suggestions on how to get more linearised plasmid? OR on using infusion specifically, (pOPINS) plasmid?
You can try the following reaction:
6ul linear plasmid
1ul mutant gene
2ul 5x Infusion Enzyme premix
1ul water
then transforms into commercial highly competent cells (preferably neb-10-beta) use 1-2 ul per 40 ul of cells. Hope this helps.
your essentially adding the same total amount as the previous student when you do it this way (since you are doubling the volume of vector).
I'm not familiar with the Bioline Genomic DNA kit, but I wouldn't use a genomic DNA kit to isolate plasmid. Use a proper plasmid kit, or do an old-fashioned Birnboim-Doly alkaline lysis preparation (see Molecular Cloning, a laboratory manual).
It's also possible to increase plasmid yield by treating your culture with chloramphenicol.
For your already isolated plasmid I can recommend DNA purification kits which are able to concentrate your product (but you will lose some of it), if the volume is limited.
Otherwise use Plasmid isolation, linearize plasmid and purify it. You can easily achievy more then 30 ng/ul just using kits. Also read section DNA recovery in your kit's manual.
Are you sure that you have plasmid not genomic DNA? It would explain why it didnt work too.
How do your amplified linearized plasmid look on the gel? In GENERAL, any of the generic column purification kits( Promega/Sigma/Molbi etc) should give you at LEAST 50ng/ml of purified product, assuming a standardized reaction volume at cycle number. What volume are you setting up for your PCR for the linearized plasmid and/or the insert. I would recommend at least 25ul, or ideally 50, especially if you have you to do further DPN treatment of your linearized plasmid.
Hussam Ali Mohammed How curious to see your completely vacuous answer garnered 2 recommendations. If you are seriously just farming for "recommendations" to increase your score, you are doing a major disservice to this wonderful resource.
Hi, Gemma Marshall.
As Dr Andrew Jenkins said, the genome kit is not for plasmid purification. You can see it here at the bioline catalog ("https://www.bioline.com/downloads/dl/file/id/1197/nucleic_acid_isolation_guide.pdf").
So, probably, you will get better results using a proper designed kit for your goal.
Another thing is, although I did not use infusion system, The amount of plasmid and PCR product need to be calculated properly by using molar ratio of both of them as below:
Typically, In-Fusion Cloning reactions work well with 100 ng of a 4- to 5-kb linearized vector plus 50 ng of a 1-kb PCR product. If your vector and PCR product differ greatly in size (e.g., you are using a 12-kb vector and 60-bp insert), the molar ratio calculator may recommend using less than 50 ng of insert.
Check any available calculators like the takara one:
https://www.takarabio.com/learning-centers/cloning/in-fusion-cloning-tools/in-fusion-molar-ratio-calculator
Hope it helps you!
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