Sentelab:Notebook/gene synthesis/2010/09/18

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(=1st PCR product purification by PCR purification kit)
Current revision (10:27, 17 September 2010) (view source)
(=1st PCR product purification by PCR purification kit)
 
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#5 volumes of buffer PB added to volume of the PCR reaction and mixed
#5 volumes of buffer PB added to volume of the PCR reaction and mixed
#A QIAquick column is plaed ina provided 2 ml collection tube  
#A QIAquick column is plaed ina provided 2 ml collection tube  
-
#To bind DNA, the sample is applied to the QIAquick  
+
#To bind DNA, the sample is applied to the QIAquick column and centrifuged for 45 sec. Flow trough is discarded and QIAquick column is placed back into the same tube.
-
column and centrifuged for 45 sec. Flow trough is discarded and  
+
#To wash 0,75 ml buffer PE is added to the QIAquick column and centrifuged for 45 sec .Flow through is discaded and QIAquick column is placed back into the same tube.
-
QIAquick column is placed back into the same tube.
+
-
#To wash 0,75 ml buffer PE is added to the QIAquick column and centrifuged for
+
-
45 sec .Flow through is discaded and QIAquick column is placed back into the same tube.
+
#The column is centrifuged in a 2ml collection tube for 1min
#The column is centrifuged in a 2ml collection tube for 1min
#Each QIA quick column is placed in a clean 1.5 ml microcentrifuge tube.
#Each QIA quick column is placed in a clean 1.5 ml microcentrifuge tube.

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=1st PCR product purification by PCR purification kit

  1. 5 volumes of buffer PB added to volume of the PCR reaction and mixed
  2. A QIAquick column is plaed ina provided 2 ml collection tube
  3. To bind DNA, the sample is applied to the QIAquick column and centrifuged for 45 sec. Flow trough is discarded and QIAquick column is placed back into the same tube.
  4. To wash 0,75 ml buffer PE is added to the QIAquick column and centrifuged for 45 sec .Flow through is discaded and QIAquick column is placed back into the same tube.
  5. The column is centrifuged in a 2ml collection tube for 1min
  6. Each QIA quick column is placed in a clean 1.5 ml microcentrifuge tube.
  7. The elute DNA for increased DNA concentration 30 ul EB buffer added to the center of the QIAquick membrane let the column stand for 1 min and than centrifuged.
  • Nano drop results
  1. 1-11 - gfp oligos : 100,3 ng/uL
  2. 12-22 gfp oligos : 89,7 ng/uL



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