Electrophoresis
Nucleic Acid Electrophoresis

Heteroduplex- and SSCP-Analysis

In heteroduplex analysis, the conformational properties of the double stranded molecules are used to distinguish different base pairing (i.e. mutations), based on the differing mobilities.
Single Stranded Conformational Polymorphism (SSCP) refers to the differing migration properties of single stranded molecules affected by variations in base sequence. 

SERDOGEL SSCP 2 x Concentrate

R: 45 - 46 - 22 - 43 - 48/20/21/22 - 62 -
S: 53 - 20 - 24 - 37/39 - 45 - 60
[T] T 

Mut.  Cat.: 2, Canc.Cat. 2, Rep.Cat. 3  • MAK/TRK: Krebserzeugend, Abschnitt III, Gruppe A2
EG-Index-No.: 616-003-00-0   • GGVSE/ADR: 6.1/25c
IATA: 6.1 III UN 2810  • WGK 3
HS-No. 38220000  • CAS-No. [79-06-1]  • Mr 71.1

SERDOGEL SSCP is a gel concentrate which has been designed for the use in DNA electrophoresis, especially for DNA conformational structure analysis (SSCP and heteroduplex electrophoresis). SERDOGEL SSCP is also suitable for the calculation of molecular weight (separation of DNA markers, DNA fragments etc.). SERDOGEL SSCP is supplied as a 2 X concentrate. It can be used like a acrylamide solution showing 35 %T. Handling of SERDOGEL SSCP is similar to common acrylamide solutions.
Specifications (PDF) available !

Ordering Information
Cat.-No.   Amount
10690.02 250 ml
10690.03 500 ml

 

Handling of SERDOGEL SSCP 2 x Concentrate

SERDOGEL SSCP represents the gel matrix only. Other components like buffer, APS, TEMED, urea (if required) must be added before preparing the gel (see protocol below). SERDOGEL SSCP has been aliquoted under an inert atmosphere. The shelf life of an unopened bottle is 
12 months if stored at 4 °C.

For preparing a gel, degas the solution (3 minutes) before adding APS and TEMED. Polymerization time is comparable with normal acrylamide/bis-solutions (about 1 hour). Gels can be cast down to a dilution of 0.5 X (which corresponds to a 1:4 dilution of the SERDOGEL SSCP 2x concentrate).Further components for gel preparation, e.g. urea, glycerol,can be added. All common detection methods are applicable, i.e. silver staining and chemiluminescence. Detection can be performed as well using SERDOGEL SSCP.

Safety precautions:
SERDOGEL SSCP contains acrylamide, which is a neurotoxicand mutagenic substance. Please do wear appropiate clothing and gloves. Even polymerized gel matrices still contain traces of acrylamide monomers.

 

Protocols for preparing gels with SERDOGEL SSCP for heteroduplex- and SSCP-analysis

For 100 ml gel solution, the amounts of required components are listed below:

Component Heteroduplex-analysis
1.0 x SERDOGEL SSCP		 SSCP-analysis
0.5 x SERDOGEL SSCP
SERDOGEL SSCP
2x concentrate		 50 ml 25 ml
TBE buffer, 10x
(Cat. No. 42557)		 6 ml 6 ml
Urea1)
(Cat. No. 24524)		 15 g ­
Glycerol2)  
75 %		 ­ 13 ml
Water
(demin.)		 ad 100 ml ad 100 ml
TEMED
(Cat. No. 35925)		 40 µl 40 µl
APS (10% solution
(Cat. No. 13375)		 400 µl 400 µl

1) At this concentration, the effect of urea is not denaturant rather than improving band sharpness.
2) Addition of 10 % glycerol is optional (this corresponds to 11.5 ml of 87 % glycerol p.a., cat.no. 23176)

Casting

  1. Prepare a mixture of all components listed except forAPS and TEMED in a beaker or a vacuum flask.
  2. Use a magnetic stirring bar for mixing the components and degas the solution for 3 minutes under vacuum.
  3. Add APS and TEMED to the reaction mixture and stirgently by shaking the solution (prevent air bubbles).Cast the gel. The use of BlueSlick (Cat. No. 42500) for pretreatment of glass plates is recommended, which functions as a repellent to remove the gel more easily from the glass plate after electrophoresis.
  4. Polymerize the gel for at least 1 hour at room temperature (or over night).

Electrophoresis

A pre-run of the gel before electrophoretical sample preparation is not necessary. Standard conditions for electrophoretical separation are given below. It must be mentioned, that band resolution is not only a matter of electrophoretical parameters, but is also affected by individual sample preparation. For best results the following parameters have to be adjusted in each individual case.

Parameters: Adjust the power supply to a constant voltage of 800 V. This corresponds approximately to a value of 7 mA at the beginning of the run (gel formate 33 x 40cm; thickness 0.35 mm). Other formates can be run with 20 V/cm at room temperature. In SSCP analysis, gels canalso be run with a permanent value of 6-8 W.

 

Time for electrophoretical separation (at room temperature):

Base pairs Volt hours Hours
ca. 170 10.000 12.5
ca. 350 14.000 17.5
ca. 700 20.000 24.0

Dye-marker position: Xylene cyanol corresponds with 270 bp, bromphenol blue with 80 bp.

 

Detection procedure: Silver staining1)
1) Bassam et al., Anal. Biochem., 196, 80-83, 1991

a) Stock solutions:

10 % acetic acid
20 % silver nitrate solution (SERVA Cat. No. 35110 in water (demin.))
10 mg/ml sodiumthiosulfate solution (Na2S2O23 x 5 H2O)

b) Procedure:

Step Time Reagents
1) Fixation 2 x 10 min 10 % acetic acid
2) Rinsing 3 x 2 min water (demin.)
3) Staining 1 x 30 min 0.5 ml of 20 % silver nitrate solution (= 0.1%)
150 µl formaldehyde 
(HCHO 37 %)
ad 100 ml with water (demin.)
4) Rinsing 1 x 10 sec water (demin.)
5) Developing 2 to 5 min at 4 to 10 °C !!
3 % sodium carbonate solution
150 µl formaldehyde
20 µl of sodiumthiosulfate stock solution (10 mg/ml)
ad 100 ml with water (demin.)
6) Fixation 1 x 5 min 10 % acetic acid
(see step 1)
7) Rinsing 2 x 5 min water (demin.)
8) Equilibration
for storage		 1 x 5 min 1 % glycerol in water (demin.)


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