I am trying to get a gene knock-in in rice by using biolistic transformation. Basically, I will shoot a CRISPR vector + high-amount of donor (1800 kb, obtained through PCR) in rice callus.
To do that, I need a very high amount of PCR-amplified donor: 2400 ng/uL. I will need a total of 5 uL every time I transform.
I have no idea how to get such a high concentration! If I put together a lot of PCR and then purify them all together, the column of the purification kit will get saturated .
If I put together a lot of already purified PCR and then dry them to increase the concentration, I will get a lot of salts together with my DNA, which will not help my transformation.
What can I do?
If your amplimer is a single clean band then desalting after mixing is cheap and easy with sephadex bead in a pasteur pipette sealed with cotton wool or glass fibre. Choose a grade of sephadex that includes the primers but excludes the pcr product.The salts are included in the beads and the dna completely excluded so just collect the early eluate . you should be able to desalt 1/3 column volume of pcr mix each time. Or even pool the pcrs and ethanol precipitate then do a single desalting step or dialyse to remove salts
If it's a single, bright band then you could also use the ExoSAPit reagent to clean up. You only need 1 microliter to remove excess primers and unused dNTPs from a 10-20 microliter reaction. No salts are included since it's an enzymatic cleanup.
You could potentially increase the concentration by letting the cleaned samples evaporate down in volume.
Good luck!
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