I am trying to insert a fragment in pHisGb1, but after sending the miniprep for sequencing, find out the vector is empty. Now how can I improve my ligation?
First you could calculate the ammount of free ends you have per DNA mass, then try to put more free ends of your insert than those you have of your vector. that will improve your ligation greatly.
Second, I don't know what conditions are you using, but in my case letting the ligation at 4º C overnight or even over weekend gave me better results than room temperature ligation.
Finally, are you using DNA comming directly from a digestion? because if that is the case, maybe you are not digesting completly the vector, and I't be great to run it in a gel and purifing the DNA band of the linear vector and using it for your ligation.
I did ligation reaction at room temperature for 2 hours. After digestion I incubate that at 37c overnight, then dephosphorylate the vectors, then did PCR purification. Then used that sample for ligation.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis
- genetic techniques
- Sequencing