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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3.1/2.3 Appendix 3


TITLE: PREPARATION OF ANALYTICAL GELS AND ELECTROPHORESIS BUFFER


PROCEDURE

  1. Clean the gel casting tray with distilled or deionised water and seal across the ends with masking tape. Place the tray onto a levelled surface.

The following are added to a 500ml pyrex conical flask:

  • Agarose Type 1 (Sigma) 2.1g or DNA grade (Biograd)
  • 10 x TBE 30ml
  • Distilled water 270ml
  1. Bring the agarose etc to boiling point and continue to boil gently until all the agarose is dissolved. Alternatively bring to boiling point in a microwave (set a high 4 min). The agarose is cooled to 55o-58oC by standing at room temperature or swirling the flask under a running tap.
  2. Add 30ml ethidium bromide (10mg/ml) and mix by swirling (10ml / 100ml of gel).
  3. Pour the agarose into the gel tray with the gel comb in place. Any air bubbles can be removed with a fine pipette tip.
  4. The gel should be allowed to set for 30min. In rooms with strong drafts cling film should be placed over the top of the gel tray to prevent the agarose surface hardening.
  5. When the agarose is set the gel comb is carefully removed place in a gel tank. Submerge the gel in fresh 1 x TBE containing ethidium bromide (100ml of 10mg/ml stock solution per 1l of buffer). The wells should be at the negative end of the tank.
  6. Gels should be used on the same day that they are prepared but in exceptional cases they may be stored overnight in 1 x TBE + ethidium bromide before use.

Note: All batch numbers of reagents used should be recorded on the gel blot log.

Safety note:

Gloves should be worn at all stages of agarose gel preparation. Special care should be taken when handling ethidium bromide solution and the stock solution should be kept in a light proof bottle and clearly labelled.

REAGENT

10XTBE

  • TRIZMA BASE 162g
  • Boric Acid (Analar) 46.3g
  • EDTA 9.5g

Make up to 800ml with distilled water

Adjust pH to 8.8 (with conc hydrochloric acid)

Make up to 1l with distilled water

Check conductivity of a 1:100 dilution in distilled water (200-230ms).

Autoclave and store at room temperature for up to six months.
 


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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