I am trying to do DNA shuffling on a ~500bp gene. After DNaseI treatment, I need to separate and extract ~50-150bp DNA fragments for further reassembly. Methods reported in the literature are mostly based on dialysis. It will be helpful if someone guide me with any other rapid methods available for this application?
Start with the LARGE scale PCR. ( 1ml or more) Do DNAse treatment on conc. sample in 4-8 reactions series: stop using EDTA after 5s, 10s etc.
Run them all on the 2% gel side by side (avoid long runs,
to maintain agarose/DNA)
Choose the ones that give you a lot of product in the required range.
Cut the gel slices and gel extract (don't forget i-PrOH)
keep in mind that shuffling is overrated,
PCRing several clones together under error prone
conditions is almost as good as shuffling.
Thanks Misha Soskine for the reply.
Actually, my question is about extraction method. Generally, short length (50-100bp) DNA extraction from agarose-gel is not recommended using commonly used gel-extraction kits.
that's why "large scale PCR"
you'll need it to overcome the low yield.
How do you save this problem that "Any suggestions for simple separation of DNA shuffling products?" ? can you give me some advice ? and how can I get enough DNA fragment size for DNA fragment?
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