Agarose Gell Electrophoresis

University
Swansea University
Course
PM134 | Microbiology
Pages

2

Academic year

2023
Author

Sanjeev K Ganesh
Views

0

EXPERIMENT NO: 2 3 DATE 10.10.2022 AGAROSE GEL ELECTROPHORESIS AIM DNA check run by electrophoresis PRINCIPLE Gel electrophoresis separates DNA fragments by size in a solid support medium such as an agarose get sample (ONA) are pipetted into the sample wells, followed by the tively charged DNA to migrate (electrophorese) towards application of an electric current which causes the nega the anodal, positive (+ve) end. The rate of migration is proportional to size: smaller fragments move more quickly and wind up at the bottom of the gel DNA is visualized by including in the gel an interealating dye, ethidium bromide DNA fragments take up the dye as they migrate through the get Illumi nation with ultranolet light causes the intercalated dye to fluoresce. The larger fragments fluorisce more intensely. Although each of the fragments of a single Class of molecul include less mass of DNA, take up less dye, and therefore is present in equimolar proportions, the smaller fragma known size can be run simultaneously and used to esti fluoresce less intensely A ladder set of DNA fragments of mate the sizes of the other unknown fragments. MATERIALS REQUIRED Agarose TIBE buffer Gel casting tray, comb, power pack sample DNA Loading dye Sterile microtips Ethidium bronnicle UV transilluminator/ge documentation system PROCEDURE I Seal the two ends of gel casting tray using cellophane 2. Boil it till the agarose gets dissolved completely Prepare 30 mL of 0.8% agarose solution using TBC buffer 3.

I A. Add 3,111 of Et br (ttrudium bromide) to the agarose solution 5. cool the solution (50-60°C) pour into get casting tray with comb planed 6 Allow it to solidify 7 Remove the comb and cellophane once its solidified 8 Mount the get in electrophoretic tank and fill the tank with TBC buffer till gel sinks 9. Mix 10ml of DNA sample with 2 ill gel loading age in glass slide 10 Load the samples into wells and perform electrophonesis at 50V OBSERVATION Separ ate DNA bands are observed under U Vtraneillurring an a the bands are orange in colour. ator RESULTS presence Orange coloured bands of DNA indicates the for of genomic DNA of E. coli