If you have any questions, let me know.  good luck
 
DNA EXTRACTION Bees & Mites H. Ferguson, Nov. 1994 D. Sammataro
NOT FOR PUBLICATION, INFORMATION ONLY  Feb. 1996.
 
1.  Freeze sample with little or no liquid. Grind insect in glass or
plastic grinder with Longmire's lysis buffer. If using bees, use one
thorax. If mites, 1-100 mites will give good results. Final Vol =89 400ul.
Note: You can also microwave sample, dry or in 10ul Longmire's for 1-4 min,
grind in lysis buffer.
2.  Pipet 1:100 dilution of Proteinase K  (10 mg/ml), approximately 4ul
into each sample and shake the tubes.
3. Incubate at 65C for 1-2 hours.
4. Add 56 ul 8M KoAc and put on ice for 30m to bring the cellular debris dow=
n.
5. Centrifuge, making sure that the tubes are balanced, and spin them down
for 10 m.
6. Using disposable plastic transfer pipette, remove TOP (clear) portion
and squirt it into a newly labeled tube.
7. Add 0.5/ vol isopropanol and let stand at RT for 8 min (apprx. 250=B5l).
8.  Shake, then centrifuge again for 8 m.
9.  Decant and wash precipitate with 70% cold ethanol (-20C); centrifuge 10
min. Dry overnight or with vacuum dryer.
10. Resuspend in 50-100ul TE O/N.  Heat for 5 min at 65C to kill DNAase.
Note: Procedure produces lots of RNA, so treat with RNAase if this is a
problem.
Longmire's lysis buffer:  RT storage of DNA material (Blood, mites, bees etc=
.)
=46inal [Conc]:           Use to make:
100 mM Tris pH 8.0      50ml 2M Tris pH8.0
100 mM EDTA             200ml 0.5M EDTA
10 mM NaCl              2ml 5M NaCl
0.5% SDS                50ml 10% SDS
0.2g Na Azide.          0.2g Na Azide
                698ml dH20
 
Synergel (1.6%):
0.5x TBE        Synergel        Agarose
100 ml          0.55g           0.7g
200ml           1.1g            1.4g
250             1.38g           1.75g
 
NOT FOR PUBLICATION
 
Date: Tue, 6 Jun 1995 18:09:33 -0500 (CDT)
To: Diana Sammataro <[log in to unmask]>
cc: Clay Comstock <[log in to unmask]> Clay Comstock
Dickinson State University
Dickinson, ND 58601
Subject: Re: RAPDs
1. Grind spiders in a 1.5 tube using disposible transfer pipets.
2. Add 500ul SDS Extraction Buffer
3. Add 20ul Protinase K (20mg/ml)
4. Incubate at 55-60C for 2-5hrs
5. Add 500ul of PC(25:25)
6. Centrifuge 3min
7. Extract top layer and add to new tube containing 500ul of chloroform
8. Centrifuge 3min
9. Extract top layer and add to new tube containing 1000ul of 95% EtOH.
Estimate the volume of EtOH and DNA and add 1/10 NaOac
10. Place tubes in freezer overnight
11. Centrifuge at 4C for 15min
12. Decant 95% EtOH and NaOac
13. Add 500ul of 70% EtOH and resuspend pellet
14. Centrifuge at 4C for 15min
15. Decant 70% EtOH
16. Air dry pellet
17. Resuspend pellet in 500ul of TE Buffer
 
 
 
 
Diana Sammataro, Ph.D.
The Ohio State University, OARDC/ Dept. Entomology
Extension Bee Laboratory, 1680 Madison Avenue
Wooster, OH 44691
Phone: (216) 263 3684  Fax: (216) 262 2720
Email: [log in to unmask]