Template:SBB-Protocols PCR3

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(SOEing PCR)
Current revision (17:24, 22 February 2010) (view source)
 
Line 1: Line 1:
===SOEing PCR===
===SOEing PCR===
*Set up PCR reactions according to your construction file as normal 33uL reactions as described in '''Cloning by PCR'''
*Set up PCR reactions according to your construction file as normal 33uL reactions as described in '''Cloning by PCR'''
-
*For each PCR, load 8uL of PCR product premixed with 1uL of loading buffer in a single well of a 1% agarose gel
+
*For each PCR, load 6uL of PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel
*Cut out the bands, put them into a single 1.5mL microcentrifuge tube
*Cut out the bands, put them into a single 1.5mL microcentrifuge tube
*Add 650uL of ADB Buffer
*Add 650uL of ADB Buffer

Current revision

SOEing PCR

  • Set up PCR reactions according to your construction file as normal 33uL reactions as described in Cloning by PCR
  • For each PCR, load 6uL of PCR product premixed with 4uL of loading buffer in a single well of a 1% agarose gel
  • Cut out the bands, put them into a single 1.5mL microcentrifuge tube
  • Add 650uL of ADB Buffer
  • Proceed with the Zymo Gel Purification procedure
  • Elute the DNA in 50uL of water
  • Set up your second round of PCR as a normal 33uL reaction using the eluted mixture of fragments as template
Personal tools