geneclen try this?

ewingd at omrf.uokhsc.edu ewingd at omrf.uokhsc.edu
Tue Feb 15 12:07:32 EST 1994


The following is the protocol our lab uses for Geneclean.  Much of it is
from the procedure included in the kit.

Start at step4 if DNA is not in agarose.

1. Use flanking ethidium bromide stain to excise desired DNA from low 
melting point agarose (Seaplaque [ or Newsieve for small]).

2. Water bath to 50C. (45-55C) Get ice.

3. Weigh on tared weighing paper.  Aliquot and slice-up .4g or less to 
ufuge tube(s). (We use a coverslip for cutting.)

4. Add 2.5 (2-3) volume NaI solution (kept at 4C)

5. Place in waterbath. 2min.  Is it completely dissolved?  Repeat as 
necessary up to 15min.

6. Completely resuspend Glassmilk (Easier if stored on its side.)

7. Rule of thumb: Add 5uL Glassmilk to 5ug or less DNA and add 
additional 1uL for each 0.5ugDNA above 5ug.

8. Vortex.  Put on ice 5min.

9. Pellet 5sec after reach high at 4C. (Eppendorf) Tap hard on kimwipe to
remove liquid.

10. Keep on ice.  Wash 3x with 200uL 'NEW'wash (-20C) by resuspending as 
add solution. Completely resuspend using pipet tip. __ On last spin, tap
out solution then turn tube 180degrees in ufuge and use sequencing tip to
get out all of solution.

11. Elute DNA by Slowly adding and resuspending with e.g., 5uL TE 
(Total volume in steps 11&13 will equal 2x amount of glassmilk used) without
losing glassmilk to tip, i.e., add 3uL stir then add remaining at the top of
the suspension. Incubate 3min at 50C.

12. Pellet for 30s on high at room templ and pull off liquid to labeled utube
using sequencing tip.  Save tip for next step. If multiple tubes were used
for the same DNA, DNA can now be pooled.

13. Elute again with e.g. 6uL TE pulling off with tip used in step 12. (A 
third time does not add more than 1% to the total DNA yield and is usually
not done.) A small amount of glassmilk in final sample is not a problem.

14. Store at -20C.

Not resuspending in NEW washes reduces yield by more than 50%.

D. Ewing   ...stranger than fiction.




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