Team:HokkaidoU Japan/Notebook/August10

From 2010.igem.org

(Difference between revisions)
(PCR Reaction System)
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* All but [[Team:HokkaidoU_Japan/Material_And_Methods#Materials_And_Methods|3-1E]] was moved to new plates
* All but [[Team:HokkaidoU_Japan/Material_And_Methods#Materials_And_Methods|3-1E]] was moved to new plates
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==PCR Reaction System==
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==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System==
Reaction system was prepared for 3 parts, 245 uL in total
Reaction system was prepared for 3 parts, 245 uL in total
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* Template DNA length is calculated by adding prefix and suffix length
* Template DNA length is calculated by adding prefix and suffix length
** Biobrick Length + Prefix length + Suffix Length
** Biobrick Length + Prefix length + Suffix Length
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* Because Mini prep of 3-1E failed, it amplified by PCR
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* Because [[Team:HokkaidoU_Japan/Protocols|Mini prep]] of 3-1E failed, it amplified by PCR
==PCR program==
==PCR program==

Revision as of 08:20, 2 October 2010

Single Colony Isolation

  • Observed if any colonies were made
    • Could not see 3-1E
  • Number of other colonies on other plates was also very small
    • Mistake in protocol is inferred
    • Precipitation of cells maybe at fault
  • All but 3-1E was moved to new plates

PCR Reaction System

Reaction system was prepared for 3 parts, 245 uL in total

Reagent Amount
Autoclaved DW 165 uL
10x PCR buffer 25 uL
2 mM dNTPs 25 uL
25 mM MgSO4 15 uL
EX-F primer 5 uL
PS-R primer 5 uL
KOD plus Neo 5 uL
Total 245 uL
  • 49 uL of reaction solution was added to each of 4 tubes and after DNA template was added
  • Length of each template is listed int the table below
Tube Biobrick Length
1 2-24G(sender) 847 bp
2 1-2M(RBS) 61 bp
3 1-23L(terminator) 178 bp
4 3-1E(heat sensor) 984 bp
  • Template DNA length is calculated by adding prefix and suffix length
    • Biobrick Length + Prefix length + Suffix Length
  • Because Mini prep of 3-1E failed, it amplified by PCR

PCR program

Predenature 94℃ 2 min
Denature 98℃ 10 sec
Extension 68℃ 30 sec
Hold 4℃
  • 30 cycles
  • KOD potency is 30 sec/kbp, so 30 sec for1cycle

Electrophoresis

Electrophoresis of amplified BioBricks
  • Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
  • Used marker pUC1d19/Hinf1
  • Added 20 uL of EtBr to 1/2 TBE
Lane DNA Length of DNA
1 pUC1d19/Hinf1
2 2-24G(sender) 847 bp
3 1-2M(RBS) 61 bp
4 1-23L(terminator) 178 bp
5 3-1E(heat sensor) 984 bp