Gel purification ligation problem

Jose Sierra jsierra at udec.cl
Mon Jul 30 00:52:34 EST 2001


Hi,
    I recommended de next protocol (is very succeful for me):

   1) use agarose low melting (GibcoBRL: 15517-022)
   2) Cut your band of interest (low exposicion at UV).
   3) Put in eppendorf and melting agarose to 60 celcius dergree.
   4) Complete the volume to 500 microliter with TE pH 8
   5)  Add 1 vol of Phenol (saturated in Tris) pH 8.
   6) Vortex with energy for 30 seconds and centrifuge for 3 minute at 12000
or Max RCF
   7) Take the aquose phase. Repeat step 5 one more time but using 100  
microliters of TE pH 8
   8) Add 1 vol of Phenol:Chloroform (1:1) at your aquouse phase and repeat
step 6.
   9) Take the aquose phase.
  10) Add 1 vol of Chloroform:Isoamylic (24:1) at your aquouse phase and
repeat step 6.
  11) Add 1/10 volumen of Sodium acetate 3M pH 5.2. and 2.5 volumen of
Absolute ethanol (-20 degree celcius). Vortex for 10 seconds and -20 degree
for 30 minutes.
  12) Centrifuge for 10 minutes at 4 celcius degree.
  13) Wash your pellet with 500 microliters of Ethanol 70 percent (eliminate
salts).
  14) resuspend the pellet in TE pH 8.0 ( depending of the size of your
fragment it can be 20 or 30 microliters).
15) Store at -20 degree.
16) Use before 3 days for ligation. The nuclease ares very actives yet you
are using EDTA in the TE buffer for inactivate it.

      Good Luck,


  15)
   


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