Hi every one,
I used the GenepHlow Gel/PCR kit (Geneaid) for DNA gel extraction. But I got low yield of my product. I've tried the suggestion on the protocol but it doesn't work. Does any one have suggestion for improving the product yield for this kit?
Thanks!
you should tell us that if you are getting a good band of not but still.
I think you should do following things:
1. if you are not getting sharp and bright bands and If you have more sample remaining with you than you can load more amount sample in the well in this way you will get sharp band and more amount of DNA on gel extraction .be careful and do not overfill your wells this will give smear instead of sharp band. if you cannot do this than
2.You should divide your elution buffer say according to protocol you need 50 microliter elution buffer so you could divide this and perform elution by using 25 microliter volume of your elution buffer than spin and again add another 25 microliter of your elution buffer. if this is not effective than
3. Use less amount of elution buffer in last step of your extraction.
Hope this will work
All the Best
Dear Hao Han Pang!
If DNA fragment is >2 kb, prewarm Elution Buffer to 65 °C before applying to column and incubate column for 3-4 min at room temperature before centrifugation.
It worked for me, I hope you will too!
Good job!
I agree with the comments given above. How much did you load on the gel for extraction? Were you able to cut all portions of the gel containing your DNA band? How did you determine the yield? Did you measure concentration by using a Nanodrop? Did you run purified DNA on agarose gel? Did you try a nuclear-free water instead of the elution buffer?
Thank you for all your answers.
For detail condition, I used sharper gel for DNA extraction. The size of my DNAis about 800 bp, and I load 1 microgram DNA sample per well without overloading. I think the band was sharp enough. I tested EB buffer and nuclear free water for elution, water would be better for elution. I got recovery lower then 10%, and I thing using lower column of water was a better condition.
Thanks for all your advises, I will try different condition next time.
Note all above important comment and I added that after first 1 min centrifuge and beffor washing step add 100 microliter binding buffer to center of column to keep more your DNA
All the above comment are really helpful, in my case i usually elute 30 micro liter of DNA which is relatively do no dilute the DNA as well as before elution i incubate the kit with elute/distilled water for 3/4 minutes in room temperature.
Hope that might help you
you can re-pass the sample from column to increase the yield. It will reduce the concentration but increase the yield.
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