Team:HokkaidoU Japan/Notebook/August10

From 2010.igem.org

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Line 8: Line 8:
** Mistake in protocol is inferred
** Mistake in protocol is inferred
** Precipitation of cells maybe at fault
** Precipitation of cells maybe at fault
-
* All but [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|3-1E]] was moved to new plates
+
* All but [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]] was moved to new plates
==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System==
==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System==
Line 54: Line 54:
|-
|-
| 1
| 1
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|2-24G]](sender)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]](sender)
| 847 bp
| 847 bp
|-
|-
| 2
| 2
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-2M]](RBS)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]](RBS)
| 61 bp
| 61 bp
|-
|-
| 3
| 3
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-23L]](terminator)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]](terminator)
| 178 bp
| 178 bp
|-
|-
| 4
| 4
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|3-1E]](heat sensor)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]](heat sensor)
| 984 bp
| 984 bp
|}
|}
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|}
|}
* 30 cycles
* 30 cycles
-
* KOD potency is 30 sec/kbp, so 30 sec for1cycle
+
* KOD potency is 30 sec/kbp, so 30 sec for 1 cycle
==Electrophoresis==
==Electrophoresis==
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* Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
* Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
-
* Used marker [http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC1d19/''Hin''f1]
+
* Used marker [http://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''f1]
* Added 20 uL of EtBr to 1/2 TBE
* Added 20 uL of EtBr to 1/2 TBE
Line 104: Line 104:
|-
|-
| 1
| 1
-
| pUC1d19/''Hin''f1
+
| pUC119/''Hin''f1
|  
|  
|-
|-
| 2
| 2
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|2-24G]](sender)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]](sender)
| 847 bp
| 847 bp
|-
|-
| 3
| 3
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-2M]](RBS)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]](RBS)
| 61 bp
| 61 bp
|-
|-
| 4
| 4
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-23L]](terminator)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]](terminator)
| 178 bp
| 178 bp
|-
|-
| 5
| 5
-
| [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|3-1E]](heat sensor)
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]](heat sensor)
| 984 bp
| 984 bp
|}
|}

Latest revision as of 07:14, 27 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 for 1 cycle

Electrophoresis

Electrophoresis of amplified BioBricks
  • Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
  • Used marker pUC119/Hinf1
  • Added 20 uL of EtBr to 1/2 TBE
Lane DNA Length of DNA
1 pUC119/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