DNA isolation from Drosophila larvae - (Sep/18/2006 )
Hello All,
Kindly guide me for Isolation of good quality DNA from Drosophila larvae....and PLz tell me detailed protocol if possible Thanks.
with regards
Mahen
Kindly guide me for Isolation of good quality DNA from Drosophila larvae....and PLz tell me detailed protocol if possible Thanks.
with regards
Mahen
For fly larvae you can use normal phenol-chloroform DNA extraction as described by Sambrook (adjust volumina), salting out protocols (if not too much slime is produced by the larvae) or kits (genomic insect DNA kits are available, they work). Some protocols improved for insects/Drosophila are available in the net (google) e.g. http://www.imba.oeaw.ac.at/fileadmin/files...20drosophila%22
which ever way you do decide to use for DNA extraction and purification, I would recommend using liquid nitrogen for the homogenisation step. Add liquid nitrogen, turn the tissue (eg fish, fly, baby mouse) into brittle glass and grind with a motar & pestle. It is simple and works very fast., even with large quantities of tissue.
This method is ever better for extracting protein and RNA from tissues. In fact, it is the best method for RNA extractrion from tissues since the RNAse inhibitors will hit the biggest surface area at once after you have ground up the frozen tissue. Good suggestion!
LTR
This method is ever better for extracting protein and RNA from tissues. In fact, it is the best method for RNA extractrion from tissues since the RNAse inhibitors will hit the biggest surface area at once after you have ground up the frozen tissue. Good suggestion!
LTR
Hi !
I am having trouble finding motar and pestle to grind tissue. Could any one please suggest from which company can i get them. I require fully glazed material with a capacity to hold 100ml. it is a small one.
Most which i have seen in fischer are one with unglazed ends. ie the end of the pestle and the center of the mortar
thank you in advance
hi there,
See You can use fresh tissue. Use Liquid N2 for extraction and use protocol mentioned at www.bioprotocol.com for Drosophila DNA extration it will work.. IF Liquid N2 is not there just freeze the tissue in -20 or -80 fro 20-30mn and use it....it will work.. for pricipitaion of DNA some time 95% Alchol will give good results that 100%....
Many users say this is the best fly DNA extraction protocol: http://www.aquaplasmid.com/Protocol.html#fly
AquaGenomic Drosophila Protocol (Courtesy of Kelly Beumer at The University of Utah)
1. Add 50 ul AquaGenomic solution to 1 fly in 1.5 ml microfuge tube.
2. Homogenize with Kontes disposable pestle, either by hand or with the Kontes pellet pestle motor for 5-10 sec. Up to 48 preps at a time can be done. Leave the homogenized flies on the desk until all flies have been homogenized.
3. Incubate at 60˚ C for 4 min.
4. Vigorously vortex each tube 20-40 sec.
5. Centrifuge sample at max speed (16000 rpm) for 4 min.
6. Transfer supernatant to new tube containing 0.7 volume isopropanol. Mix.
7. Centrifuge at max speed for 4 min; decant supernatant.
8. Rinse with ~500 ul 70% ethanol by shooting the ethanol solution from a squeeze bottle just below the rim of the tube. Decant ethanol and pipet off remaining solution.
9. Allow tubes to dry ~15 min at room temperature.
10. Add 30 ul 10 mM Tris, TE, or ddH2O to DNA. Leave on bench overnight, vortex, or heat at 60˚ C for one hour to resuspend.
11. Store at either 4˚ or –20˚ C
0.5 ul of this solution is sufficient for a PCR reaction.