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

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) here

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

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

Sadao Ota 15:03, 25 February 2009 (EST)

Ligation of EcoRI/BamHI digests

• Set up the following reaction:

 6.5uL ddH2O
 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
 1uL Vector digest
 1uL Insert digest
 0.5uL T4 DNA Ligase

• Pound upside down on the bench to mix
• Give it a quick spin to send it back to the bottom of the tube
• Incubate on the benchtop for 30min
• Put on ice and proceed to the transformation

I used gel to separate the DNA I need I also used digested DNA without gelation to do heat shock.

2nd PCR cloning did not work. So, PCRing again...