Low insert and vector concentration - (May/01/2007 )

inspite of the very high concentration i had at the begning, my vector and insert became ~5ng/µl after gel putification. Do you guys think i can proceed with this concentration for ligation or need to concentrate it before further? I performed gel purification using Invitrogen Pure link kit, in its procedure it recommends elution of final DNA with 50µl of warm TE; which resulted in the dilution of my final product.

I have another Kit Invitec-eurobio which could be used for concentration of final products and PCR purification. Do you think it will have an impact on my final product if i go through two different purification kits? like losing DNA extrimities? or anything that might affect ligation reaction?
I really appreciate your answer it means a lot me
Thank you
Eminov

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usually for ligation experiment i start from 5 ug DNA (i know its too much) and i never tried with 5 ng.

my lowest conc was 100 ng/ul.

you can try EASY TRAP ver. 2 from TAKARA to purify DNA from gel. it gives me always a better recovery of DNA

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I routinely ligate with 5 ng (2.6 fmoles) of vector. 1 uL of a 5 uL ligation reaction is transformed with 25 uL of chemically competent cells and diluted with 680 uL of SOC and 30 uL of this per plate generates 300-500 colonies per plate.

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I would like to try your way, isn't the 680µL SOC a lot though for one hr incubation? what about 250µl?

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If I used 250 µL of SOC, I would have 770 to 1280 colonies/plate.
Reducing the plate inoculums below 30 µL makes it more difficult to spread the colonies evenly.
It is difficult to pick single colonies from plates that are so crowded.

If your ligation and transformation efficiencies are lower than mine, then using 250 µL of SOC may be beneficial. Historically, 9 volumes of SOC is recommended, which would be 225 µL to a 25 µL transformation.

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I would say, forget about the concentration of your insert and vector. Just proceed with your ligation. You will never know if it is working. For your information, I never bother about my vector and insert concentration nowadays. As long as when I checked the purified products on gel and they are still visible, I will proceed with ligation.

You can always try different ratio for your ligation.

Good luck.

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Thank you all, first i will go on with Tfitzwater idea and see if it works with my ligatyion and transformation! one thing more i want to ask is if you guys ever tried triple digestion all enzymes been having the same buffer? like buffer 4 of new england biolabs?
Thanks again i will let you know the result of my trials!

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yes i tried triple digestion with same buffer, works well

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