We have a bacterial strain with multiple plasmids, I m interested in one plasmid which around 45kb, no selection marker is available for this plasmid thus not conjugated nor transformed so the only way we are trying is cutting out of the gel but the yield after purification with kit is still low, any recommendations?
Which kit are you using? One obvious thing you could do is load a lot more of the sample so you have a thicker bank (to cut out) and more DNA to perform the kit protocol on.
I am using Promega kit for PCR/gel purification, I load the max. i can but the yield is still low (10ng/microlitre) and i need at least 30ng/microlitre to be sent for sequencing.
I assumed as much. You could do two things.
1. I have really good experience with QIAquick gel extraction kit. I used it all through my PhD and never had issues (link below). The only problem is that the kit purifies DNA from 70 bp-10 kb and might not be ideal for your 45 kb. But I looked up the promega kits and seems they do the same. You could maybe try the QIAquick kit.
https://www.qiagen.com/ca/shop/sample-technologies/dna/dna-clean-up/qiaquick-gel-extraction-kit/#orderinginformation
2. If you are loading max into a well already, try loading max amount of your sample into multiple wells. Then cut out all the relevant bands and purify them in the same tube if you can (starting concentration for the kit is usually 10 ug and so you might not be able to process all the bands together). Also try reducing the elution volume. This way you should have a really concentrated DNA solution which you could dilute if needed.
I found these two links which could help you further.
https://bitesizebio.com/13506/10-tips-for-better-dna-gel-extraction-results/
https://bitesizebio.com/13508/5-more-tips-for-dna-gel-extraction/
I would really try that and get back to you! Thanks, really appreciate it.
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