SBB09Ntbk-Sadao Ota

Sadao Ota 15:03, 9 February 2009 (EST)

My project is SBB09_20154, to make ag43 Display (short) ag43 Display (long) systems. Designed a oligo M10034 to make Autotransporter protein with promotors and so on (corresponding to amino acids 708-1017). Also, designed another oligo M10035 to make Autotransporter protein only (corresponding to amino acids 517-1017).

• Construction files are M10034 M10035
• Oligonucleotide sequences are Oso001, Oso002, Oso003

Sadao Ota 15:03, 16 February 2009 (EST)

Do PCR with the combination of Oso001&Oso002, and Oso002&Oso003.

Dilute oligonucleotides to be 10 uM

• Add 1 ul of oligo dilution to 9 ul of ddH20
• Mix and spin

Cloning PCR, (products are 937bp & 1600bp, )

• 24 uL water
• 3.3 uL Expand 10x Buffer 2
• 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
• 1 uL Oligo Odvs007F (10uM)
• 1 uL Oligo Odvs008rR (10uM)
• 0.5 uL Expand Polymerase 1
• 0.5 uL Template DNA E.coli 0157:H7
• Run this PCR on program 55

Sadao Ota 15:03, 23 February 2009 (EST)

Clean up PCR products for gelation analysis

Sadao Ota 15:03, 23 February 2009 (EST)

Clean up PCR products, Digest, and Clean up again.

Regular Zymo Cleanup (removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction)

1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
2. Transfer into the Zymo column (small clear guys)
3. spin through, discard waste.
4. Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
5. spin through, discard waste.
6. Add 200 uL of PE or Zymo Wash buffer
7. spin through, discard waste.
8. spin for 90 seconds, full speed to dry.
9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Load to Gel to perform gelation analysis (dead or alive)

• mix the eluted DNAs (3 ul) and loading dye (2 ul)

EcoRI/BamHI Digest of PCR Products (my PCR products are large enough, so no need to modificate the process)

• Set up the following reaction:

 8uL of eluted PCR product
 1uL of NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI

• Incubate at 37 degrees on the thermocycler for 1hr
• Clean Up again as described before.

not yet

• Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point