Sero: p30 Crossover Electrophoresis for Semen

Transcription

1 Safety Principle Equipment and supplies SAFETY WARNING Electrophoretic procedures have electrical hazards associated with them. Therefore, at minimum, the presence of two analysts is required during these procedures. The protein p3 is semen specific. This electrophoretic procedure identifies p3. The identification of p3 in a biological stain confirms the presence of semen. This procedure uses the following laboratory equipment and supplies: electrophoresis tank electrophoresis power supply analytical balance ph meter oven set at approximately 6C Bunsen burner or heating plate glass or plastic support, approximately 7.5 cm x 5 cm appropriate glassware pipet pipet tips microcentrifuge tubes gel bond filter paper blotting paper weight (optional). Page 1 of 5

2 , Continued Buffer This procedure uses the following buffer: Tank and Gel Buffer (1 L) 25.2 g Trizma Base 2.5 g EDTA, free acid 1.9 g boric Acid Combine the above chemicals in approximately 8 ml of deionized water. When chemicals are dissolved bring to final volume of 1 liter. Check the ph and adjust to 9.1 using 2% w/v Sodium Hydroxide. Alternative Preparation: Tank and Gel Buffer: (5L) Dilute the contents of the P3 TANK AND GEL BUFFER pre-made solution (comprised of Tris, Boric Acid, EDTA, and NaOH) in 4.5 L of purified water. Label container with the pre-made solution lot number, expiration date, preparation date and preparer s initials. Store buffer solution at 5C ± 5C. Page 2 of 5

3 , Continued Reagents This procedure uses the following reagents: 2% w/v Sodium Hydroxide Dissolve 2 grams of sodium hydroxide in deionized water to bring to a volume of 1 ml. 1 M NaCl Dissolve grams of NaCl in deionized water to bring to a volume of 1 liter. Stain Solution Dissolve.2 grams of Brilliant Blue in 1 ml of Destain Solution. Destain Solution Mix the following: methanol/acetic acid/deionized water (5/1/5). SERI p3 Antiserum Page 3 of 5

4 , Continued Support medium Standards and controls Quality control Dissolve, using heat,.1 grams of agarose (SERI type E25 or agarose of similar EEO) in 1 ml of Gel Buffer. The following standards and controls must be used in each test: SERI semen standard diluted 1:2 and 1:4 in deionized water saline or water blank. New lots of buffer are tested for quality during the analysis of casework. The analyst cannot test more than one new lot of buffer in a single electrophoretic run. If problems with the standards are noted, the new lot of buffer is discarded and re-made. When a new lot of SERI p3 antiserum is reconstituted, a p3 crossover is run against the known semen standard. Typical results will show a visible precipitin band out to the 1:32 dilution of the semen standard. Affix the dried gel to the Crossover Electrophoresis Worksheet. A record of the test and the dried gel is kept in the Standards/Controls Quality Control Log Book located in the Serology/DNA Laboratory. When a new lot of SERI semen standard is reconstituted, a p3 crossover is run using 1:2 and 1:4 dilutions of the semen standard against p3 antiserum. Affix the dried gel to the Crossover Electrophoresis Worksheet. A record of the test and the dried gel is kept in the Standards/Controls Quality Control Log Book located in the Serology/DNA Laboratory. NOTE: Newly reconstituted antisera and standards with the same lot numbers as those previously tested do not need to be re-tested. Page 4 of 5

5 , Continued Sample preparation Procedure Interpretation References Extract the stain cutting (approximately 1 cm x 1 cm) or swab (approximately one quarter) in 5 l of saline for a minimum of 3 minutes. Use the following procedure to perform p3 crossover electrophoresis. Step Action 1 Pour the support medium on the hydrophilic side of the gel bond, using a glass or plastic plate for support. 2 Punch wells. 3 Load 6 l of p3 antiserum into the anodic wells and 6 l of sample, control or standard into the cathodic wells. 4 Run gel for a minimum of 2 minutes at 12 volts. 5 Wash gel overnight in 1 M NaCl. 6 Rinse in deionized water, cover with filter paper (and a weight, if desired) to remove water, and dry at approximately 6C. 7 Stain the dried gel for approximately 5 minutes with Stain Solution. 8 Destain the dried gel in Destain Solution until the background is reduced sufficiently to see the resulting precipitin bands. 9 Affix the stained gel and record the results on the Crossover Electrophoresis Worksheet. The presence of a stained precipitin band between opposing wells is a positive (+) test for the presence of p3. The absence of a precipitin band indicates a negative (-) reaction for the presence of p3. The following reference was used in the development of this procedure. Methods Manual, Serological Research Institute. Page 5 of 5