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Ligation 5 way and beyond

Posted by perneseblue, 27 August 2010 · 732 views

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.
  • 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




Wish you a good luck .
multiway ligation is gonna be one of the major techniques i have to deal with all the way through my thesis, and they look like , if i may say, pretty hard ones. first one is ligation that has to be done to clone a 7377 bp fragment into a 3000 bp vector. to increase the efficiency I have treated the PCR product with taq DNA polymerase to make sure that the majority of fragments have got the A in their end. then the reaction mix has been incubated in different conditions:
1- 4 c for an overnight period
2- 22.5 c for half an hour and then 16 c for an overnight period
at the end of incubation I have tried both deactivating the ligase by keeping the reaction mix in 65 c for 20 min and not going through the deactivation step.
finally i have transformed the competent bacteria with the transformation reaction.
the molar ratios I have used are 1:1 and 1:2.
i have screened the colonies by both blue/white screening and antibiotic resistance. then i have performed colony pcr for unlimited number of colonies( occasionally i have checked blue colonies in addition to white colonies). I have had no luck till now, even absolutely white colonies don't have the insert as they are suppose to.
Are there any other ways of increasing the efficiency of ligation, because obviously this step is the hold back here.
tnx
PS: I have tried different vectors for T/A cloning step.

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