Hello all,
I will do oligonucleotides annealing for the first time which will be later used for cloning without any more modification. Can any of you kindly suggest if I should purify the annealed DNA from ssDNA? Is it a necessary step to reduce background in the cloning? If yes, please suggest how. Do you think if I run them in agarose gel, I would see any difference?
Thanks in advance.
I did some shRNA cloning before, directly using the annealing DNAs without purification, and it works well. here is a protocol from online:
http://rnai.genmed.sinica.edu.tw/file/protocol/11_shRNA%20construction_V3.pdf
If you are trying to retain the double stranded duplex and get rid of the SS oligo then you could try exonuclease1 digestion then agarose gel purification
No it is not necessary and you would actually lose the majority of your product if you use column based purification as they are designed to remove the low size DNA.
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