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Sticky and blunt ligation issue


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#1 ramblingrat

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Posted 27 November 2013 - 08:43 AM

Hi everyone,

 

I want to clone a 1300 PCR fragment into a 8000bp plasmid using NruI (blunt end) and HindIII (sticky), but I didn't get any colonies.

Here's what I did:

Being limited with potential sites, I am using NruI (no other choice) and HindIII. So I did PCR with restriction sites and 5' overhangs, which gave bands as expected. Then I digested my PCR product and vectors first with HindIII (Buffer NEB2), gel purified everything before second digest with NruI (Buffer NEB3). Ran that on gel and saw the bands of the fragments I cut out of the backbone as expected, gel-purified insert and backbone again before ligation. So far I think everything worked.

Then I performed ligation with standard T4 DNA ligase in a vector:insert molar ratio of 1:2, 100ng in total at 16C overnight. Used 2ul of ligation for transformation with TOP10 and plated plenty of it.

 

I think most likely the issue is the ligation, and I was wondering if there is any advice what to try first to improve it. Would you just try different molar ratios at the same time? Any particular issues for mixed sticky and blunt ligation? Would dephosphorylation help?

 

Many thanks for any help!!

 

R.



#2 phage434

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Posted 27 November 2013 - 09:30 AM

It is very rare to have "no other choice." Can you explain why NruI is the only choice? Blunt end ligations often have difficulty with standard ligase buffer. You should try "quick ligase" buffer, since you have gotten this far along this path. If that doesn't work, I would switch strategies to a different enzyme avoiding blunt ligations.



#3 ramblingrat

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Posted 27 November 2013 - 11:20 AM

It's no other choice because there is no other unique restriction site at this site of my plasmid where I want to introduce my insert. So it either works or I can't do what I want, the plasmid is some new fancy stuff. 

I already e-mailed NEB about different buffers, NruI seems to have issues with that. However, before I bury my plan I was wondering if trying different ratios and DNA amount or other advice would also make sense.

 

Therefore, hoping for further advice... :o)



#4 phage434

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Posted 27 November 2013 - 01:16 PM

You can ALWAYS PCR your plasmid to add whatever restriction sites you want, wherever you want them. Just design two primers which amplify the entire plasmid, then add the correct 5' restriction sites, and some junk bases 5' of the new restriction site.  Done.



#5 thefallengrace

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Posted 28 November 2013 - 07:00 AM

@ramblingrat for blunt end, u need PEG which commonly added to quick ligase, perhaps u can try quick ligase, and have after digestion and purification, have u double check both digested plasmid and insert? sometimes they "lost" during purification. When doing your transformation, have made controls? how does the controls turn out?

@phage434 how do i amplify the whole plasmid? can i used stand taq or do i need to use long range taq? 



#6 phage434

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Posted 28 November 2013 - 07:10 AM

You can amplify with almost any PCR enzyme. Fidelity of the enzyme is less important than you might expect, since only the plasmids that have correct origins and correct selection genes will transform.



#7 ramblingrat

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Posted 04 December 2013 - 10:43 AM

Hi guys,

 

Thanks so much for the valuable advice! After some mourning I finally got the plasmid sequence from the company, so I might get back to the advice of adding restriction sites. Thanks for that!

In the meantime I got a Gibson assembly kit - trying that at the moment, but also still with obstacles ;).

 

R.






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