SBB09Ntbk-Sadao Ota: Difference between revisions
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==[[User:Sadao Ota|Sadao Ota]] 15:03, | ==[[User: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). | 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 [[SBB09_Construct3 | M10034]][[SBB09_Construct3 | M10035]] | |||
* Oligonucleotide sequences are [[SBB09_Oligos | Oso001, Oso002, Oso003]] | |||
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==[[User:Sadao Ota|Sadao Ota]] 15:03, 16 February 2009 (EST)== | |||
<pre> | |||
Do PCR with the combination of Oso001&Oso002, and Oso002&Oso003. | |||
</pre> | |||
=== === | === === | ||
* Construction file to be found [[SBB09_Construct3 | M10034]][[SBB09_Construct3 | M10035]] | * Construction file to be found [[SBB09_Construct3 | M10034]][[SBB09_Construct3 | M10035]] | ||
* Oligonucleotide sequences to be found [[SBB09_Oligos | Oso001, Oso002, Oso003]] | * Oligonucleotide sequences to be found [[SBB09_Oligos | Oso001, Oso002, Oso003]] | ||
'''Cloning PCR''' | |||
* 24 uL water | |||
* 3.3 uL Expand 10x Buffer 2 | |||
* 3.3 uL 10x dNTPs (2 microM in each; 0.2 nM 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 | |||
Revision as of 23:02, 23 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.
- Construction file to be found M10034 M10035
- Oligonucleotide sequences to be found Oso001, Oso002, Oso003
Cloning PCR
- 24 uL water
- 3.3 uL Expand 10x Buffer 2
- 3.3 uL 10x dNTPs (2 microM in each; 0.2 nM 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
Cloning by PCR This is the basic PCR method to amplify your DNA from a plasmid or genomic DNA sample using the Expand polymerase. You also use this protocol for an EIPCR reaction. The oligo concentrations in your stocks should be 100uM. You use them at 10uM in this protocol. So, you first need to make an oligo dilution of:
9uL Water 1uL 100uM oligo
You can throw away the remainder of the diluted oligo when you are done, but hold onto your stock tube! Set up the following reaction in a PCR tube: 24uL ddH2O 3.3uL 10x Expand Buffer "2" 3.3uL dNTPs (2mM in each) 1uL Oligo 1, 10uM 1uL Oligo 2, 10uM 0.5uL Expand polymerase "1" 0.5uL Template DNA The Expand temperature programs are a little complicated, so I won’t write them out here. They all involve 2 distinct cycling phases—the first 10 cycles have shorter extensions than the latter 20 cycles. You choose which one you want based on the length of your predicted product. If it is under 2kb, use 2K55. If it is between 2kb and 4kb, use 4K55. If it is over 4kb, use 8K55. In each case, start with the "55" version of the program. If you get no product or poor yield of product, repeat the same PCR reaction as two different variants, both with the “45” program. The first reaction will be the same composition as the first. For the second one, don’t add 3.3 uL of the water and instead use the volume for 3.3 uL of DMSO (dimethylsulfoxide). If these PCRs fail as well, well you'd either redesign, use a different template source, or give up.