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?
Cloning produced inserts of unexpected size and insert with insertion of a few b
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