Conformation Sensitive Gel Electrophoresis (CSGE) Protocol

Primers should be designed so that PCR products have at least 40 base pairs of unimportant sequence at each end, because mutations cannot be reliably detected near the ends.  The maximum length is about 500 base pairs.  Thus, for long exons, design PCR products that overlap by about 100 base pairs.
One plate of each pair can be siliconized.

20X TTE buffer (1 L):

3M Tris base 592 ml
0.5M EDTA 8 ml
Taurine  71.3 g
adjust pH to 9.0, filter and store at room temperature.

6X Loading dye:

0.25% Bromophenol Blue
0.25% Xylene Cyanol FF
30% Glycerol
Water
 
1) PCR:  Prepare at least 10 ul each PCR product in a 96 well plate.

2) Make gel:

100 ml                                                (1 gel)

20X TTE                                             2.5 ml

Acrylamide 40%                                37.5 ml
1,4-Bis(acryloyl)piperazine (BAP)     0.15 g in 1 ml water (prepared fresh)
Formamide                                         15 ml
Ethylene glycol                                  10 ml
Water                                                 33 ml
AMPS                                                 0.1 g in 1 ml water
TEMED                                              70 ul

Don't insert the comb fully, but just enough to fully form the wells.  Clamp the plates over the comb to prevent a thin layer of gel polymerizing between the comb and plates.  Let gel set >45 minutes.

3) Prepare for loading:  Put 2 ul water in tubes, and add 3 ul PCR products.  Denature:  95¢XC 5mins -> room temperature 30mins.  Add 1 ul 6X loading dye to PCR products.   Pre-run gel:  25W, 30mins.  Gel should not be hot.

4) Load:  Wash wells.  Load DNA ladder asymmetrically to help identify position of lanes.  Load 3 ul sample per well.  May load up to 3 samples per lane by running gel 45mins at 40W between loads.

5) Run:  40W, ~9 hours for 400 bp DNA, starting from first loading of gel.

 
6) Stain gel:  Transfer gel underwater to 3MM paper and apply SYBR Gold solution (2 ul/20 mL water) for a few minutes.  Observe position of bands with hand held UV light, then cut out that portion of the gel, and cut in half down the middle.  Further stain with 5 ml each half gel for a few minutes if necessary.

7) Photograph bands:  Place gel side down on UV transilluminator.  Wet paper and peel off to leave gel on transilluminator.  Photograph.

Last updated: 24Sep2000
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