AlanFung:Protocol/Agarose Gel Synthesis: Difference between revisions

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==Protocol==
==Protocol==
1-Determine the concentration of Agarose to be used  
*Determine the concentration of Agarose to be used
 
                                                                  (1 Tray)
             DNA Size                            5 - 10 kb                          0.2 - 1 kb  
             DNA Size                            5 - 10 kb                          0.2 - 1 kb  
   Concentration of Agarose                        0.7 %                                2 %  
   Concentration of Agarose                        0.7 %                                2 %  
Line 34: Line 34:
     Amount of SYBR Safe                            8uL                                  8uL  
     Amount of SYBR Safe                            8uL                                  8uL  
Note: Scale up or down accordingly  
Note: Scale up or down accordingly  
 
*Weigh out the required amount of Agarose with a scale
*2-Weigh out the required amount of Agarose with a scale and measure the required volume of
*Measure the required volume of TBE buffer using a graduated cylinder  
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
*Caution: Hot Agarose solution should be handled very carefully.   
*Repeat mixing and heating step until all gel powder are dissolved and the solution appears to be clear
*3-Transfer the Agarose powder into a 250mL Erlenmeyer flask and add in the TBE buffer, mix
*Let cool for a while and add in the required amount of SYBR Safe, swirl flask to mix the dye
by swirling the flask and microwave for 30s, repeat mixing and heating step until all gel
*Cast gel into tray with the required comb set in the tray
powder are dissolved and the solution appears to be clear.
*Let cool

Latest revision as of 22:51, 22 June 2009

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