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 Cleaned up the PCR reaction.

Regular Zymo Cleanup

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

#Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
#Transfer into the Zymo column (small clear guys)
#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, use the same volume of water as the volume of the original reaction

Ran on gel to check size/ presence of EaeA gene

February 25, 2009

Regular Zymo Cleanup

The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

#Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
#Transfer into the Zymo column (small clear guys)
#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, use the same volume of water as the volume of the original reaction

During this purification I put my reaction tube in the centrifuge unlabelled and it got confused with Nihkeil. We reran our samples side by side on a gel, and found which sample was my EaeA part.

Electrophoresis

2ul EaeA PCR sample
1ul Dye

Aluminum Binding Protein gene part construction

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