SBB09Ntbk-Sadao Ota: Difference between revisions
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'''Cloning PCR, (products are 937bp & 1600bp, )''' | '''Cloning PCR, (products are 937bp & 1600bp, )''' | ||
* 24 uL water | ##* 24 uL water | ||
* 3.3 uL Expand 10x Buffer 2 | * 3.3 uL Expand 10x Buffer 2 | ||
* 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) | * 3.3 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) | ||
| Line 26: | Line 26: | ||
* 0.5 uL Expand Polymerase 1 | * 0.5 uL Expand Polymerase 1 | ||
* 0.5 uL Template DNA E.coli 0157:H7 | * 0.5 uL Template DNA E.coli 0157:H7 | ||
## Run this PCR on program 55 | |||
Revision as of 23:13, 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.
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 to perform gelation analysis
Regular Zymo Cleanup (removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction)
- Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
- Transfer into the Zymo column (small clear guys)
- 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 tube, use the same volume of water as the volume of the original reaction
- 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
