SBB09Ntbk-Roger Lowe

EeaA Intimin gene part construction

February 18, 2009


Created cloning solution for EaeA Intimin gene using buffer, primers, expand polymerase, and template DNA.

*Make 100uM stocks of primers, this is the concentration used directly for the reaction
*Prepare the following reaction:
  29 uL water
  5 uL Expand 10x Buffer 2
  5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
  5 uL Oligo 1 (100uM)
  5 uL Oligo 2 (100uM)
  0.75 uL Expand Polymerase 1
February 23, 2009



Small-Frag Zymo Cleanup
After performing Klenow extension for EaeA Intimin gene, I performed Zymo Cleanup.
The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for fragments smaller than 300bp.  It also will remove the buffer and restriction enzymes from a restriction digest reaction.
#Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
#Transfer into the Zymo column (small clear guys)
#Add 500uL of Ethanol and pipette up and down to mix
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
#spin through, discard waste.
#Add 200 uL of PE or Zymo Wash buffer
#spin through, discard waste.
#spin for 90 seconds, full speed to dry.
#elute with water into a fresh Eppendorf tube
Now to be cyclized 


Created solution for Klenow (wobble) extension for aluminum binding protein, now being cyclized. 


I then continued to digest the EaeA Intimin gene.

EcoRI/BamHI Digest of Wobble Products
For wobble products, you will digest the entire extension reaction-worth of DNA Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.
 50uL eluted DNA
 5.7uL NEB Buffer 2
 1uL EcoRI
 1uL BamHI
    * Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
    * Incubate the reaction at 37 degrees on the thermocycler
    * Proceed to another Zymo small fragment cleanup