I've been trying to shuffle a 3kb gene using a method by Lorimer modified from Stemmer's protocol that uses MnCl2 instead of MgCl2. I purified the <50bp fragments by gel fltration but could not obtain a distinct band after reassembly and PCR amplification. I'm wondering if the gene is to big for this method or if the fragment size is too small. Does anyone have experience with shuffling homologous genes of this size? Any advice? Thanks!
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