Novel Application of Reversed Gel Electrophoresis: Reuse of DNA Molecular Weight Marker (100 bp DNA ladder)

Published: 22-07-2017| Version 1 | DOI: 10.17632/gn2h7bnjwh.1
Contributors:
Suhasa G,
Savithri Bhat

Description

100 bp DNA ladder is reused here for one time: The well corresponding to resolved DNA ladder is sealed with molten agarose. The resolved 100 bp DNA ladder is made to converge to a single band by reversing the electrode polarity or by facing the wells in gel towards the anode. The gel slice containing the DNA ladder is removed using scalpel or any other suitable device very carefully so that a thin rectangular gel piece containing the ladder is obtained. The gel slice or gel plug can be stored in water or in buffer at 4C in refrigerator. For reuse the gel plug is placed in a fresh well of agarose gel and the well is sealed with agarose. The test DNA samples are loaded in other wells. The electrophoresis again resolves DNA ladder from gel plug. The prior art internet search reveals commercially available low melting agarose gel plugs (eg., InCert agarose gel plugs sold by Lonza) containing large DNA fragments such as lambda DNA ladder that are used in Pulsed Field Gel Electrophoresis (PFGE). We have designed methods using reversed gel electrophoresis that favourably helps in reuse of resolved 100 bp DNA ladder.

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Steps to reproduce

1. Resolving 100 bp DNA ladder in 3% agarose (Seakem LE agarose from Lonza was used) gel and 0.5X TAE. 2. Sealing the corresponding well (from where the ladder got resolved) using molten 3% agarose. 3. Interchanging (Reversing) the wires connecting the platinum electrodes. 4. Carrying-out reversed electrophoresis for ~ 20 minutes. 5. Placing the gel in 0.5 ug/ml ethidium bromide. 6. Continuing the reversed gel electrophoresis until the DNA ladder appears in sealed agarose well. 7. Removing the gel piece containing 100 bp DNA ladder using scalpel, etc. 8. Storage of the removed gel plug in refrigerator until next use . 9. Placing the gel plug in fresh well of agarose gel and sealing the well using molten agarose without producing any air bubbles. Loading test DNA in other wells representing different lanes. 10. Carrying out normal gel electrophoresis resolves DNA ladder from sealed gel plug.