RapidGel XL Protocol
100ml RapidGel XL 40% stock solution available from Amersham Biosciences
Catalogue Number US75863.
Gels
RapidGel XL Solution 40%
Formamide
Urea
10x Long Read TBE
18mOhm water
DMSO
TEMED
10% APS (Fresh)
66cm/0.25mm
5.8ml
21 g
5 ml
30 ml
500l
75l
350l
66cm/0.2mm *
5.8ml
2.5ml
21 g
5 ml
30 ml
75l
350l
Long Read 10x TBE
Tris Base
162.0 g
(1340 mM)
Boric Acid
27.5 g
(450 mM)
EDTA (Na2, 2xH20)
9.3 g
(25 mM)
Water
add to give 1 L
pH of the 1xTBE should be between 8.3 and 8.7 at 500C (I have never checked this,
but at room temperature the 10x TBE is approximately pH 9.0).
Method
* I only have experience of the 0.2mm formulation.
1. Weigh out the urea in a 250ml beaker. Add the 40% RapidGel stock solution,
water and formamide. Stir at room temperature until the urea is dissolved and the
solution has equilibrated to room temperature. (Avoid heating the solution).
2. Add 2g Amberlite resin (IRN-150L), and continue stirring for 7 mins.
3. Pipette 5ml of long read 10x TBE into a 0.2m filter unit – pull through the filter
just prior to adding the acrylamide/Amberlite solution to avoid deionisation of
the buffer by the Amberlite. (I use water vacuum rather than a pump).
4. Filter/degas the acrylamide solution through filter.
5. Remove the top of the filter unit. Add the freshly made APS and mix by swirling.
6. Add the TEMED and mix by swirling.
7. Pour and leave for a minimum of 4 hrs before using – should begin setting
approximately 10-15 min after addition of TEMED.
I run the gel on the LI-COR using the standard 66cm settings.