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I need to clone in a 30kb DNA fragment into my plasmid (~7kb).

Working with such a large fragment I have run into a few problems. The first was purifying it from the PCR mix used to amplify it out of its template, I achieved thsi with ethanol precipitation however I am unsure if thsi removes dNTPs from the PCR mix?

The second is ligating it into my plasmid. The fragment is flanked with EagI sites, however after digestion I cannot purify the digested fragment from the end cut fragments(due to its size).

I have two questions, firstly will the dNTPs remaining from the ethanol precipitation affect digestion and ligation reactions. Secondly will the end cut fragments affect ligation reactions.

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    $\begingroup$ Is your plasmid capable of handling such a huge insert? Most of the commonly used plasmids are not. $\endgroup$
    – Chris ♦
    Commented Jan 17, 2022 at 19:17
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    $\begingroup$ Welcome to the site. Can you please edit in some more information (the more the better for troubleshooting) - how big are your fragments from the EagI site digestion? How are you trying to separate the digested from undigested? Have you looked at things like Bacterial artificial chromosomes (BAC) that might be more appropriate for this size. What is the purpose of the cloning - expression, (cloning) library prep...? $\endgroup$
    – bob1
    Commented Jan 17, 2022 at 19:27
  • $\begingroup$ Hello, Yeah so my plasmid is a bit of a weird guy, its linear (has covalently closed ends) and has been demonstrated to be able to hold up to 40kb without too much problems. I've also further modified it to potentially make it hold more. So the plasmid (being linear) has a left arm that it 5.7kb and a right arm that is 1.1kb; these are treated with EagI to leave overhangs. The fragments I am trying to clone in is 30kb and has eagi sites at each end. $\endgroup$
    – Danyn Patel
    Commented Jan 18, 2022 at 23:15
  • $\begingroup$ So this plasmid is one which I have modified from a larger linear plasmid. The whole purpose of this is to be able to store and propagate large and complex DNA fragments without needing a very low copy number plasmid. The specific purpose of it is quite broad, and could be used for many purposes. Basically trying to demonstrate that my engineered plasmid can hold such large and complex fragments. At the moment I'm assuming the fragment is being digested completely, and I am not doing a cleanup after because they don't seem to be able to cleanup such a large fragment. $\endgroup$
    – Danyn Patel
    Commented Jan 18, 2022 at 23:16
  • $\begingroup$ The problems I'm encountering are about when I'm trying to ligate things into it, as this big 30kb fragment is hard to isolate using commercially avaliable PCR cleanup kits and so on. $\endgroup$
    – Danyn Patel
    Commented Jan 18, 2022 at 23:16

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