I am working on my cloned fragments in a low copy number vector so generally, i get low yield of my recombinant plasmids. I have two different genes in two low copy plasmids i want to perform invitro ligation of both the genes together by releasing and ligating them. But after gel purification or precipitation i am getting low DNA concentration which is not enough to perform IVT.
If you are using column purification after agarose gel separation try eluting the bound dna with hot 70C elution buffer and do this 2 times.It often increases the yield
Another strategy is to start with more molecules. Do a "midi" or "mega" prep to get overall more plasmids. Be sure to read the instructions carefully - you can overload the column if you try to use way too many cells.
You can also do an old-style boiling lysis plasmid purification. No column, the cell debris & DNA is removed as a "blob" and you precipitate out the plasmid DNA.
For running on a gel, use lab tape to cover the gel comb & make one gigantic well. Load as much plasmid DNA as physically possible and you'll get (hopefully) a gigantic bright band of DNA.
Or, there is a old (VERY dangerous if you don't use the correct PPE) method. Remove the lid from your electrophoresis box. Connect the wires directly to the electrodes. Put a UV light box under the gel. Carefully cut small hole about 1/2 way down the gel below the well you have used for your sample. Make the hole the same width as the well. Use Ethidium Bromide in the gel & the running buffer. Set up the gel electrophoresis in a dark room. Wear gloves, lab coat, face shield, etc. Run the gel and occasionally turn on the UV light to check where the band that you want is located. When it "falls" into the hole you cut, you quickly pipet up the glowing liquid. Then, you precipitate the DNA. It takes practice, but it works really well.
Good luck!
If the recommendations made by others do need seem to work, you may have to provide more information. What % agarose gel are you using? How big (e.g., in mg) are the gel excisions you are using? Did you verify that your band of interest is indeed visible (and of correct size) following gel electrophoresis? Which kit or protocol do you use for gel extraction? What is the ratio of mg of gel excised product to volume of lysis buffer? Do you ensure your gel excision equipment is clean?
Lisa Pieterse thank you for your suggestions-
I am using a 1% gel and am making preparative digestion of plasmids in 100 μL. I cut the gel ~ at 400mg, and my band of interest is visible in very good concentration after elution. However, I am losing yield, which hinders the IVT process, and my gel extraction equipment is clean and I am currently using g to p kit its an Indian kit made in Lucknow for PCR and gel purification