AlanFung:Protocol/Agarose Gel Synthesis

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Agarose_Gel_Synthesis_Protocol PDF

Agarose Gel Synthesis Protocol[edit]

Overview[edit]

*Determine the concentration of agarose to be used 
*Measurement of agarose gel and TBE buffer  
*Mix and Heat 
*Cool Down 
*Gel Staining 
*Casting 
*Storage 

Samples & Material[edit]

*Erlenmeyer Flask (250mL) 
*Graduated Cylinder 
*Mircowave 
*Gel tray 13 x 12.2cm (2 standard gel trays)  
*Gel Tray (4 micro gel trays) 
*Combs for micro gels  (6/8 teeth)  
*Standard combs  (13/26 teeth)  
*Gel casting stand for micro gels 
*Gel casting stand   
*0.5X TBE Buffer 
*Ultra Pure Agarose                                                          -Invitrogen Cat:15510-027 
*SYBR Safe DNA gel stain10,000X concentration in DMSO                        -Invitrogen Cat:S33102 


Protocol[edit]

  • Determine the concentration of Agarose to be used
                                                                  (1 Tray)
            DNA Size                            5 - 10 kb                           0.2 - 1 kb 
  Concentration of Agarose                        0.7 %                                 2 % 
      Amount of Agarose                            0.7 g                                2 g 
 Amount of 0.5X TBE Buffer                       100 mL                               100 mL 
    Amount of SYBR Safe                            8uL                                  8uL 

Note: Scale up or down accordingly

  • Weigh out the required amount of Agarose with a scale
  • Measure the required volume of TBE buffer using a graduated cylinder
Caution: Hot Agarose solution should be handled very carefully.  
  • Transfer the Agarose powder into a 250mL Erlenmeyer flask and add in the TBE buffer, mix by swirling the flask and microwave for 30s
  • Repeat mixing and heating step until all gel powder are dissolved and the solution appears to be clear
  • Let cool for a while and add in the required amount of SYBR Safe, swirl flask to mix the dye
  • Cast gel into tray with the required comb set in the tray
  • Let cool