I have my oligonucleotides synthesized on a chip.
Next, I PCR amplified restriction enzyme tails into the oligo library (for blunt-end ligation later). I have been doing multiple PCR amplification reactions in parallel to increase product for subsequent steps. Then I used Qiagen MinElute to clean up the PCR mix(es). I end up with about 50-60 ng/uL after purification.
Then, for the backbone: I restriction enzyme digested the vector of cloning interest, ran it on agarose gel, then gel purified the backbone.
For the library is where I have the most trouble. After the amplification and clean up and purification of the library, I digested it and ran it on agarose gel for gel extraction. This results in a significant reduction in DNA concentration, (50-60ng/uL down to 2ng/uL). Overall, this decreases my library complexity and amount of material I have to use for ligation and transformation.
I further need to clean the ligation reaction before electroporation/transformation, again reducing about of material and loss in library complexity.
What should I do to preserve material, library complexity and still be able to transform properly? I’ve been stuck at this step
Any advice and experience are appreciated.
Maybe you could change the clone method. I recommend you use Takara In-fusion kit or NEB Golden assembly kit. Detailed operating steps，you can reference manual.
Thank you for your response @Meiye. My oligos are 146 bps, and 171 after PCR amplification prior to digest.
I thought about maybe pooling multiple ligation reactions before doing a MinElute purification prior to transformation.
I'm not sure if there's another way
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