I had amplified my fragments with primers to give an extra base pair for the restriction site, which has been digested. furthermore, the vector has been digested with same enzyme SacI. but my fragment product shorter fragment than expected one by 400bp, I did ligation then cloned into E. coli DH5a to increase the copies. unfortunately, I did not get any growth, could someone help.
According to NEB, SacI has no detectable cutting activity if there's only 1 bp between the recognition site and the end of a fragment, and you need at least 3 bp from the end to get full cleavage: https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Also, I'm a little confused about what you mean by the fragment is 400 bp shorter than expected. Is it the PCR product shorter when run on a gel just after amplification? If it is, you might need to increase the annealing temperature, play around with additives like DMSO or redesign the primers. If it's shorter after the PCR fragment is digested, does the intended insert also contain a SacI cut site?
hi
Your problems are high.you can do :
1- check SacI site in your insert (PCR product) < Should not exist SacI site in your gene sequence >
2- check optimal antibiotic concentration in LB-Agar ( kanamycin 50ug/ml ) & optimal X-gal+IPTG
3- check transformation protocol (heat shock 42c 1min ) or (Electroporation )
good luck
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