I gel-excised my pcr products and cloned it with pGEM-T easy vector. Incubated the culture on plates at 37C for 16 hours, then 4C for 24 hours. Then, picked white colonies and miniprep, followed by EcoR1 digestion for 1.30h.
When i run my gel i found the digested bands were either ~750bp (but my inserts should be 1500bp) or had 2 bands (one with correct size and another one not). Why does it happened? I tried to re-transformed and the outcome was same!
Another case where i got a partial sequence. Based on that sequence i designed primer to perform 3' RACE (ie 1 primer based on that sequence and one "poly T primer"). When i compare the 1st partial sequence with the 2nd sequence, i found that there were many insertion of few bases in the 2nd sequence. By removing the inserted bases only it codes for the correct amino acids. Why does this happen?
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Cloning produced inserts of unexpected size and insert with insertion of a few b
Started by hianghao, Oct 30 2010 12:58 AM
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