Promega Magic Mix

Dr. C.J. Chan cchan at uk.ac.crc
Sat Sep 18 07:18:08 EST 1993


Dear Netter,

I think Promega Magic Mix is no more than Diatomaceous earth (Sigma D 5284)! I
may be wrong, if so, could somebody let me know. Anyway I am now routinely usingthis stuff in place of Promega Magic Mix and as a result saved a lot of money.

Here is the protocol,

Reagents and Material

Diatomaceous earth (Sigma D5384)
4M guanidine thiocynate (Sigma G6639), 50mM TRIS-HCl pH7.0, 20mM EDTA
80% isopropanol
acetone

Preparation of DNA binding matrix

Shake up 25 g of diatomaceous earth in 500ml of water and let it settle for 3
hours. Suck away the supernatant containing those fine particles that still
remain in suspension.

Add water to 100ml and shake to resuspend the matrix. ( 25g of diatomaceous
earth would swell up to ~50ml with water. When resuspended in 100ml volume,
the concentration is 250mg/ml) Store in 2x 50ml Falcon at room temperature.

Add 1ml of the resuspended (250mg/ml) matrix to 25ml of 4M guanidine thio-
cynate, 50mM TRIS-HCl pH7.0, 20mM EDTA. 

This is your home made Promega Magic Mix ready to go. Store in the dark at
room temperature.

Method

You can use this home made DNA binding matrix with the little plastic columns
that Promega provide or do the following.

Make up the volume of your DNA digests or PCR products to ~500ul with either
water or TE.

Add 1ml of the resuspended DNA binding matrix. Mix and leave for 1 min.

Spin 10-20 seconds. Remove supernatant carefully with pippette. The matrix is
loose at this stage and would come off the walls of eppendorfs. I normally 
remove only 1.2-1.3 mls.

Add 1ml of 80% isopropanol. Resuspend matrix gently and spin briefly again.
Pour off supernatant. (The matrix is now firmly pack and there is no danager
of it coming loose)

Wash a second time with 1ml of 80% isopropanol. Resuspend gently and spin briefly again. Pour away supernatant.

Wash a third time with 1ml of acetone. Again gently resuspending and spin briefly. Pour away supernatant. Invert the tube and leave to air dry for ~5 mins.
(The acetone wash step is important. It helps to get rid of remaining isopropanol and helps to dry the pellet quickly)

Resuspend the dried pellet in 60ul of water/TE and place in water bath for 3-5
mins to elute the DNA out.

Spin hard for 5 mins and carefully remove the supernatant. (The matrix is again
loose). Your DNA should now be in the supernatant.

Comments.

I did not invent this method. It is a modified version of Carter and Milton's
method in NAR 1993 21(4),1044.

I have not accurately determined the recovery but I think it is well over 90%.

Using Promega's minicolumn is certainly much quicker, but with a few samples
doing it in eppendorf tubes is not that painful either.



Hope this is of use to people.

Yours,

CTJ Chan
Dept of Molecular Genetics,
Institute of Child Health,
30 Guilford St.,
London WC1N 1EH
cchan at uk.ac.crc



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