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multiway ligation


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6 replies to this topic

#1 mahsa

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Posted 23 June 2010 - 03:47 AM

hey all


I am going to do a multiway ligation. the problem is the fragment size. one insert is 80 bp and the other is 7377 bp and they are gonna be cloned in pCDNA 3.1(+). I would appreciate it if you could advise me.
restriction sites on fragments are as followed:
1- 5' EcoRI 7377 XhoI 3'
2- 5' XhoI 80 EcoRI 5'
3- digestion product of pCDNA 3.1(+) using EcoRI

and the 80 bp fragment is limited in amount.
waiting here in anticipation for your reply
tnx alot
Mahsa

#2 phage434

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Posted 23 June 2010 - 05:19 AM

This will be a difficult ligation. You will have significant background from religation of the vector, and competing reactions with back to back insertions of the long and short inserts. I hope you are ready to screen many samples, because you have no selection for the proper assembly. Colony pcr with carefully selected primers is probably the easiest screening approach. Are you sure you can't move to a double digested vector, or use compatible overhang enzymes to eliminate the sites on correct ligation, and include the restriction enzymes in the ligation mix?

The ligation should be done with equimolar amounts of the two inserts and 3-5 times less molar amount of the vector, which will optimize production of your desired product.

#3 mahsa

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Posted 23 June 2010 - 06:32 AM

This will be a difficult ligation. You will have significant background from religation of the vector, and competing reactions with back to back insertions of the long and short inserts. I hope you are ready to screen many samples, because you have no selection for the proper assembly. Colony pcr with carefully selected primers is probably the easiest screening approach. Are you sure you can't move to a double digested vector, or use compatible overhang enzymes to eliminate the sites on correct ligation, and include the restriction enzymes in the ligation mix?

The ligation should be done with equimolar amounts of the two inserts and 3-5 times less molar amount of the vector, which will optimize production of your desired product.





Thanks a lot phage434
I've got no choice. have to do it exactly this way, so hope it works.


thank you again

#4 molstudent

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Posted 04 July 2010 - 10:22 AM

could you possibly put the 80bp fragment into the plasmid containing your 7377bp fragment first, then cutting out that fragment as a whole and putting it into pcDNA3.1(+)? i believe pcDNA3.1(+) is around 5400+ bps too so the ligation will be hard being that the insert is bigger than the vector

#5 mahsa

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Posted 13 July 2010 - 01:04 AM

could you possibly put the 80bp fragment into the plasmid containing your 7377bp fragment first, then cutting out that fragment as a whole and putting it into pcDNA3.1(+)? i believe pcDNA3.1(+) is around 5400+ bps too so the ligation will be hard being that the insert is bigger than the vector


tnx molstudent
i am afraid that is impossible because of the restriction enzyme recognition site design, if i do so it would be inconvenient for the rest of the process.
tnx again

#6 astartus

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Posted 13 July 2010 - 01:15 AM

I thing you could try is to add EcoRI to ligation, so that XhoI is joined first. Then you can inactivate EcoRI and do another ligation for EcoRI-sites ... maybe this increases efficiency, just an idea.

Edited by astartus, 13 July 2010 - 01:18 AM.


#7 mahsa

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Posted 18 August 2010 - 09:51 PM

I thing you could try is to add EcoRI to ligation, so that XhoI is joined first. Then you can inactivate EcoRI and do another ligation for EcoRI-sites ... maybe this increases efficiency, just an idea.

:rolleyes: , creative! a big issue with EcoRI is its star activity. although there are no restriction sites for EcoRI in fragment, the star activity can cause more damage.

tnx alot




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