Discussion
5 years ago
Nicolai Brandt
3Hello everyone! I am currently using different DNA isolation kits to extract DNA from insects. Even though I am able to successfully extract DNA I would like to maximize the yield. Do you have any tips that might help me with that even if the kits are not specifically designed for insect samples?
Experiment: DNA isolation / purification Tissue - bronchoalveolar lavage
Top comment
Firstly, you should try to macerate the sample as much as you can. Then you should incubate it in proteinase K for an extended amount of time of approximately 12 hours. Finally, for the elution, warm up the buffer to 60 degree C, use only half of the recommended amount of buffer and let the solution sit on the membrane for 10 minutes.
Answered 5 years ago
Sangeeta Kumari
3 Comments
Thank you, this was helpful!
Answered 3 years ago
Lionel Test (efty)
One of my first lab jobs was in a Drosophila lab. This may be a little old school, but we had two methods; one for single fly PCR and another for genomic harvest. Single fly PCR was done using a small pestle that fit down into a 1.5mL tube; take the fly, add some lysis buffer (proteinase K, NaOH and SDS, essentially) and grind it up. Let it sit overnight at 50C, then immerse the tube in boiling water. We would then chill it on ice, spin it down, and do PCR on the supernatant. This was good for PCR, but not much else. Genomic preps involved gathering a bunch of flies with phenotypic marker we were using, and grinding them up in a glass Dounce homogenizer with that same lysis buffer, let it go over night again, and then do phenol/chloroform extraction to get the DNA. We used this stuff for Southern blots, library preps, sequencing, etc. You could also probably use a bead homogenizer (e.g., the Omni BeadRuptor) instead of the Dounce.
Answered 4 years ago
Jerri R. Manzer
Firstly, you should try to macerate the sample as much as you can. Then you should incubate it in proteinase K for an extended amount of time of approximately 12 hours. Finally, for the elution, warm up the buffer to 60 degree C, use only half of the recommended amount of buffer and let the solution sit on the membrane for 10 minutes.
Answered 5 years ago
Sangeeta Kumari
Can you help?