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Dear Blog,
I have decided to find the limits of multi-way ligation. Thus far, I have determined that the techniques I am using gives me a reproducible limit of 6-ways. 7-way ligation occasionally works. And all attempts at 8-way ligation have ended in failure. My current screen size is 96 colonies..
At this point there are two obvious ways I could expend the number of multiway ligation.
The quality approach is more difficult, but there must be some room for improvement. I can't imagine that the methods I am using are the best possible. To this end, I have come up with a few ideas
Thus far I have not had the opportunity to see if these modification works
I have decided to find the limits of multi-way ligation. Thus far, I have determined that the techniques I am using gives me a reproducible limit of 6-ways. 7-way ligation occasionally works. And all attempts at 8-way ligation have ended in failure. My current screen size is 96 colonies..
At this point there are two obvious ways I could expend the number of multiway ligation.
- I could screen more colonies and using colony pooling, I could screen 384 colonies on a 96 well PCR plate. However this does leave the minor and tedious problem of picking 384 colonies. (ie The quantity approach)
- I could improve the efficiency and fidelity of the ligation reaction. (ie The quality approach)
The quality approach is more difficult, but there must be some room for improvement. I can't imagine that the methods I am using are the best possible. To this end, I have come up with a few ideas
- Use a ligase with increased fidelity (unable to find one).
- Heating the DNA fragment before ligation
- Dephosphorylating every other fragment.
Thus far I have not had the opportunity to see if these modification works
