Team:HokkaidoU Japan/Notebook/August10

From 2010.igem.org

(Difference between revisions)
(→PCR Reaction System)
 
(15 intermediate revisions not shown)
Line 1: Line 1:
{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
-
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August9|August 9]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August11|August 11]]</div></div>
+
<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>
==Single Colony Isolation==
==Single Colony Isolation==
* Observed if any colonies were made
* Observed if any colonies were made
-
** Could not see 3-1E
+
** Could not see [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]
* Number of other colonies on other plates was also very small
* Number of other colonies on other plates was also very small
** Mistake in protocol is inferred
** Mistake in protocol is inferred
** Precipitation of cells maybe at fault
** Precipitation of cells maybe at fault
-
* All but 3-1E was moved to new plates
+
* All but [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]] was moved to new plates
-
==シングルコロニーアイソレーション==
+
==[[Team:HokkaidoU_Japan/Protocols|PCR]] Reaction System==
-
コロニーを確認したところ，3-1Eにはコロニーが見られなかった<br>
+
-
ほかのプレートもコロニー数が少なかったため，手順に不完全なところがあったのかもしれない
+
-
* 実験中に沈殿が見られたため
+
-
** コンピテントセルの溶解が不十分だった？
+
-
** 混ぜ方が不十分だった？
+
-
* ２回目の実験で，若干急いだ感じだったので，吸着などが不十分だった？
+
-
3-1E以外を新しい培地に移した
+
-
 
+
-
==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
Line 49: Line 40:
| 5 uL
| 5 uL
|-
|-
-
|style="border-top:1px solid #000;"|'''Total'''
+
|style="border-top:1px solid #996;"|'''Total'''
-
|style="border-top:1px solid #000;"|'''245 uL'''
+
|style="border-top:1px solid #996;"|'''245 uL'''
|}
|}
Line 63: Line 54:
|-
|-
| 1
| 1
-
| 2-24G（sender）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
| 847 bp
| 847 bp
|-
|-
| 2
| 2
-
| 1-2M（RBS）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
| 61 bp
| 61 bp
|-
|-
| 3
| 3
-
| 1-23L（terminator）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
| 178 bp
| 178 bp
|-
|-
| 4
| 4
-
| 3-1E（heat sensor）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
| 984 bp
| 984 bp
|}
|}
* 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
-
* Because Mini prep of 3-1E failed, it amplified by PCR
+
* Because [[Team:HokkaidoU_Japan/Protocols|Mini prep]] of 3-1E failed, it amplified by PCR
-
==PCR反応液の調整 ==
+
==PCR program==
-
==
+
{|style="margin-left: 20px;" class="protocol"
-
今回は4パーツ増幅のためTotal 245 uL作成した．
+
-
{|style="text-align: center; margin-left:20px" class="protocol"
+
|-
|-
-
| Autoclaved DW
+
| Predenature
-
| 165 uL
+
| 94℃ 2 min
|-
|-
-
| 10x PCR buffer
+
|style="border-top:1px solid #996;"| Denature
-
| 25 uL
+
|style="border-top:1px solid #996;"| 98℃ 10 sec
|-
|-
-
| 2 mM dNTPs
+
| Extension
-
| 25 uL
+
| 68℃ 30 sec
|-
|-
-
| 25 mM MgSO<sub>4</sub>
+
|style="border-top:1px solid #996;"| Hold
-
| 15 uL
+
|style="border-top:1px solid #996;"| 4℃
-
|-
+
-
| EX-F primer
+
-
| 5 uL
+
-
|-
+
-
| PS-R primer
+
-
| 5 uL
+
-
|-
+
-
| KOD plus Neo
+
-
| 5 uL
+
-
|-
+
-
|style="border-top:1px solid #000;"|'''Total'''
+
-
|style="border-top:1px solid #000;"|'''245 uL'''
+
|}
|}
 +
* 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
-
* 反応液49 uLを４本のPCRチューブに分注し，Templateを1 uLずつ加えた
 
-
* それぞれのPCRチューブとTemplate，lengthは
 
{|border="1" style="margin-left: 20px;" class="protocol"
{|border="1" style="margin-left: 20px;" class="protocol"
 +
!Lane
 +
!DNA
 +
!Length of DNA
|-
|-
| 1
| 1
-
| 2-24G（sender）
+
| pUC119/''Hin''f1
 +
|
 +
|-
 +
| 2
 +
| [[Team:HokkaidoU_Japan/Parts#BioBricks|2-24G]]（sender）
| 847 bp
| 847 bp
|-
|-
-
| 2
+
| 3
-
| 1-2M（RBS）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-2M]]（RBS）
| 61 bp
| 61 bp
|-
|-
-
| 3
+
| 4
-
| 1-23L（terminator）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）
| 178 bp
| 178 bp
|-
|-
-
| 4
+
| 5
-
| 3-1E（heat sensor）
+
| [[Team:HokkaidoU_Japan/Parts#BioBricks|3-1E]]（heat sensor）
| 984 bp
| 984 bp
|}
|}
-
* DNA lengthはパーツ長＋プライマー(49 bp)
 
-
* 3-1EはTransformationがうまくいかなかったため
 
-
 
-
==PCR program==
 
-
{|style="margin-left: 20px;" class="protocol"
 
-
|-
 
-
| Predenature
 
-
| 94℃ 2 min
 
-
|-
 
-
|style="border-top:1px solid #996;"| Denature
 
-
|style="border-top:1px solid #996;"| 98℃ 10 sec
 
-
|-
 
-
| Extension
 
-
| 68℃ 30 sec
 
-
|-
 
-
|style="border-top:1px solid #996;"| Hold
 
-
|style="border-top:1px solid #996;"| 4℃
 
-
|}
 
-
* 30 cycles
 
-
* KOD potency is 30 sec/kbp, so 30 sec for１cycle
 
-
 
-
==電気泳動==
 
-
* 増幅したDNAを5 uLとって6xサンプルバッファー1 uLを加えた
 
-
* マーカーはpUC119/''Hin''f1を使用
 
-
* エチブロは20 uL使用
 
-
[[Image:HokkaidoU Japan 20100810a.jpg|200px|right|thumb|short discription]]
 

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"