Team:HokkaidoU Japan/Notebook/August10

From 2010.igem.org

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<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August2|August 2]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August11|August 11]]</div></div>
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==Single Colony Isolation==
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* Observed if any colonies were made
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** Could not see [[Team:HokkaidoU_Japan/Parts#BioBricks|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 [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]] was moved to new plates
 +
 +
==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System==
 +
 +
Reaction system was prepared for 3 parts, 245 uL in total
 +
 +
{|style="text-align: center; margin-left:20px" class="protocol"
 +
|-
 +
!Reagent
 +
!Amount
 +
|-
 +
| Autoclaved DW
 +
| 165 uL
 +
|-
 +
| 10x PCR buffer
 +
| 25 uL
 +
|-
 +
| 2 mM dNTPs
 +
| 25 uL
 +
|-
 +
| 25 mM MgSO<sub>4</sub>
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| 15 uL
 +
|-
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| EX-F primer
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| 5 uL
 +
|-
 +
| PS-R primer
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| 5 uL
 +
|-
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| KOD plus Neo
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| 5 uL
 +
|-
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|style="border-top:1px solid #996;"|'''Total'''
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|style="border-top:1px solid #996;"|'''245 uL'''
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|}
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 +
* 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
 +
 +
{|border="1" style="margin-left: 20px;" class="protocol"
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|-
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!Tube
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!Biobrick
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!Length
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|-
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| 1
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
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| 847 bp
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|-
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| 2
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| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
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| 61 bp
 +
|-
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| 3
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
 +
| 178 bp
 +
|-
 +
| 4
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
 +
| 984 bp
 +
|}
 +
* Template DNA length is　calculated by adding prefix and suffix length
 +
** Biobrick Length + Prefix length + Suffix Length
 +
* Because [[Team:HokkaidoU_Japan/Protocols|Mini prep]] of 3-1E failed, it amplified by PCR
 +
 +
==PCR program==
 +
{|style="margin-left: 20px;" class="protocol"
 +
|-
 +
| Predenature
 +
| 94℃ 2 min
 +
|-
 +
|style="border-top:1px solid #996;"| Denature
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|style="border-top:1px solid #996;"| 98℃ 10 sec
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|-
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| Extension
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| 68℃ 30 sec
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|-
 +
|style="border-top:1px solid #996;"| Hold
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|style="border-top:1px solid #996;"| 4℃
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|}
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* 30 cycles
 +
* KOD potency is 30 sec/kbp, so 30 sec for １ cycle
 +
 +
==Electrophoresis==
 +
[[Image:HokkaidoU Japan 20100810a.jpg|200px|right|thumb|Electrophoresis of amplified BioBricks]]
 +
 +
* Added 1 uL of 6xSample Buffer to 5 uL of amplified DNA
 +
* Used marker [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''f1]
 +
* Added 20 uL of EtBr to 1/2 TBE
 +
 +
{|border="1" style="margin-left: 20px;" class="protocol"
 +
!Lane
 +
!DNA
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!Length of DNA
 +
|-
 +
| 1
 +
| pUC119/''Hin''f1
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|
 +
|-
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| 2
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
 +
| 847 bp
 +
|-
 +
| 3
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
 +
| 61 bp
 +
|-
 +
| 4
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
 +
| 178 bp
 +
|-
 +
| 5
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
 +
| 984 bp
 +
|}

Latest revision as of 07:14, 27 October 2010

Notebook

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
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August10"