SBB09Ntbk-Sadao Ota: Difference between revisions
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'''[[SBB09_PCR Cloning | PCR Cloning]]''' | '''[[SBB09_PCR Cloning | PCR Cloning]]''' | ||
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==[[User:Sadao Ota|Sadao Ota]] 15:03, 23 February 2009 (EST)== | ==[[User:Sadao Ota|Sadao Ota]] 15:03, 23 February 2009 (EST)== | ||
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Revision as of 13:08, 25 February 2009
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.
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...