I have a 5038 bp DNA fragment I am trying to extract from an agarose gel with little success. I am running a 50 cycle PCR with HF Q5 Polymerase and get a bright band of the expected size. I have tried both the Qaigen and Monarch gel extraction kits. Monarch performs a little better, but my yield is still low (32.5ng/ul).
I have tried the following adjustments:
1. 1% and 0.7% agarose gels
2. TAE and TBE buffers
3. Centrifuge speeds between 2000-13000 rpm
4. 50C elution buffer
5. Recycling eluate up to 3X through the column.
6. 5 minute RT incubation after adding EB to the column membrane
7. Ensuring the gel is completely dissolved
How can I improve yield? Is there another method of extracting DNA from agarose gels (freezing gel)?
Thanks!
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction
More Sajid Alvi's questions See All
Similar questions and discussions