SBB09Ntbk-Roger Lowe
From OpenWetWare
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 1February 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 tubeNow 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