User:Norman Wang/DNA Isolation Via Agarose Gel Filter+Membrane Extraction
Materials
- Agarose Gel
- DNA Dye: Ethidium Bromide or SYBR Safe (a Cyanine Dye)
- Glass Based Filter
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thin filter, easy to insert, less volume, bigger chance DNA may elude the trap
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thick filter, harder to insert, bigger volume, smaller chance DNA may elude the trap
- Dialysis Tubing 3500mwco
Procedure [1]
Yield
Starting DNA quantity (total in the 10uL loaded)
- 1.0 kb: 124ng
- 1.5 kb: 122ng
- 3.0 kb: 120ng
- 6.0 kb: 48ng
Resulting DNA quantity (total in the 10uL eluted)
- 1.0 kb: ng, ng
- 1.5 kb: ng, ng
- 3.0 kb: ng, ng
- 6.0 kb: ng, ng
Seven of the eight band extraction yielded some DNA. We started with no more than 124ng of DNA in each well. Bands below 1kb were too faint to be visibly stained by SYBR Green using recommended concentration.
The experiments were performed by Megan Nakashima and Norman Wang
- Agarose Gel DNA Band Extraction Results
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Extraction of the 1kb and 1.5 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing extracted band
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Extraction of the 3kb and 6 kb band from NEB 2-log ladder, 1) separation 2) post filter insertion 3) post electrophoresis-into-filter 4) verify missing extracted band
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Extraction Yield, first row: 1kb and 1.5 kb; second row 3kb and 6kb
References
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personal communication with David Thompson of Liu Lab @ Harvard
