SSCP Protocol

MDE gel (BioWhittaker Molecular Applications) final concentrations

·        .5X MDE

·        .6X TBE

·        possible additives: 10% glycerol or 10% sucrose

Running buffer 

·        .6X TBE

Loading buffer

·        95% formamide

·        5 mM NaOH

Make up fresh gel mix for each run:

10 ml MDE gel mix

24 ml 1X  TBE

  4 ml glycerol

  2 ml ddH₂0

 40 ml total

Note:  4 g of sucrose can be used in place of glycerol.  Then add ddH_sO to obtain 40 ml. 

Add 270 ul 10% APS + 27 ul Temed to polymerize.  Let set for ½ hour.  Remove comb.  Wrap in plastic wrap.  Place in refrigerator overnight (temperature range of 3-9 ^oC).

Running the gel:

1)      Place gel rig and running buffer in refrigerator (3-9^oC) overnight.

2)      Gel will be run in refrigerator (3-9^oC).   Prerun gel for 15 m at 50 W.

3)      For single strands, mix 2 ul PCR product for each sample with 10 loading buffer. 

4)      Heat ss prep at 95^o C for 3 m.  Immediately place on ice for at least 2 m.  Load 4-6 ul.

5)      At least in the beginning, run 1 sample of non-denatured PCR product for each locus to see where ds bands fall on gel.  Dilute 39:1 with TE.  Load 5 ul + 1 ul loading buffer.

6)      At least in the beginning, use a ladder as a reference point, non-denatured.  You may need to dilute it 99:1 with TE.  Load 5 ul + 1 ul accompanying loading buffer.

7)      Run gel at  50 W for 2 hours 15 min (for first screening).  You may adjust to run longer once you see how far bands run.  My experience has been that 4 hours is the upper limit at this wattage (bands become fuzzy after 4 hours probably due to loss of conformations).  If you run for 4 hours, you might want to reduce the wattage to 40 during the last hour (to avoid fuzzy bands).

 Staining Protocol

1.2% agarose overlay with Sybr Green I Nucleic Acid Gel Stain (BMA)

For 100 ml:

-add 10 ul Sybr Green to 50 ml ddH₂O in a plastic beaker

-microwave 1.2 g agarose in 50 ml of 1X TBE in a glass beaker for 2 min.

-pour hot agarose into plastic beaker with Sybr Green solution

1) Pop open the gel plates using the flat edge of a spatula.

2) Pour overlay onto gel and let set for at least 15 min.

Peel off agarose overlay.  Use a spatula to separate edge for peeling. 

Last Updated:

02/27/03