AlanFung:Protocol/Agarose Gel Synthesis

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Agarose_Gel_Synthesis_Protocol PDF

                                               Ho Lim Fung 
                                    Agarose Gel Synthesis Protocol 
                                                Overview 
*Determine the concentration of agarose to be used 
*Measurement of agarose gel and TBE buffer  
*Mix and Heat 
*Cool Down 
*Gel Staining 
*Casting 
*Storage 
                                                 Material 
*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 

1-Determine the concentration of Agarose to be used

            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

2-Weigh out the required amount of Agarose with a scale and measure the required volume of TBE buffer using a graduated cylinder

Caution: Hot Agarose solution should be handled very carefully. 3-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.