I have been successfully restricting two plasmids with XbaI and XhoI. I add about 1ug of plasmid DNA to each reaction and have been getting the most beautiful bright bands on my gels (0.75% - See attached pic). However, when I excise those bands and attempt to extract the fragments from gel, I have been getting around 3ng/ul yield. (tried 30-50ul total elution)
What I've tried so far:
1. I have tried both the Qiagen kit and a brand new Promega Wizard kit.
2. Longer incubation times and more vortexing during gel melting step.
3. Letting tubes sit for 1 min between applying melted gel/binding buffer mix before centrifugation.
4. Extra washes, also at slower speeds (2000g for 6 mins).
5. Elution buffer/Water to elute - both warmed to 60C.
6. Allowing elution buffer/water to sit in column for 5 - 10 mins before eluting.
7. Multiple elutions have also been attempted (i.e. 2 x elutions with 25ul a piece)
I'm completely at my wits end and have no idea what else I can try, nor why this particular gel extraction is causing such a headache! Any help would be hugely appreciated!!
Thanks!!
Your not really doing anything wrong. You tend to lose quite a bit of DNA during gel extraction. Assuming a perfect condition (you getting your entire 1ug back) that will be equal to be about 33ng/ul, obviously in the lab that will not happen as some DNA will be lost during the extraction, if we take a pessimistic view and say that the efficiency is around 50% then that will drop you to approx 16.5ng/ul and even less if your extracting bigger fragments. The solution to your problem is simple, I usually simply just start out with 2ug of DNA (Do not go over 3ug though since your gel will be overloaded) and then you will end up and a decent yield. On a practical note, the yield that you have is sufficient for ligation if that is your downstream application since the transformation in E.coli can be carried out with pg amounts of DNA some of which are bound to be your proper construct.
Hi Sam Waterworth , I made so much gel extraction with the Qiagen kit and I obtain approximately the same your concentration. After this step I made the cloning or sequencing and each time I did not have problem.
Thank you so much Hanna and Giorgia! I'll try increasing the amount to 2ug as suggested and then continue on to the ligation step, even if the yield is low! I really appreciate the help :)
My lab manager swears by freezing the gel slice at -80C or -20C for about 30 mins prior to extraction. Good luck with your science!
Please check your Gel extract quality by nanodrop before proceeding with Ligation & Transformation. Most of the time with low yield there is huge 230 peak. May inhibit ligation.
Good Luck.
Hi Sam,
I had almost the same situation when I did the gel extraction last two months using Promega Wizard kit. The main problem for me is that I didn't add enough EtOH into the washing buffer. So the consequence was a lot of DNA will be washed and won't stay on the column. I have asked similar question on researchgate and someone gave out meaningful advices. Maybe you can check the answers and see if there is any possibility to meet your main problem.
By the way, even if the problem is solved, the recovery rate is still very low (20%-40% by 30µl -50 elution buffer at last step). Although the manual of the kit said it will be over 80%...😂
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