Team:HokkaidoU Japan/Notebook/August11

From 2010.igem.org

(Difference between revisions)
(→グリセロールストック作成)
 
(9 intermediate revisions not shown)
Line 13: Line 13:
==Ligation==
==Ligation==
-
===ligation用DNAの調製===
+
===DNA Preparation for Ligation===
-
* 前日にPCRした50 uLのうち電気泳動しなかった49 uLを使用した
+
* Used 49 uL of yesterdays PCR product
-
* 制限酵素処理するまえにプライマーを除去するため，ろ過を行った
+
* Removed primers via Microcon YM-10
-
** 分子量10000 Da以下を通すフィルタ（緑チューブ）を使用
+
** Added 450 uL of TE to make final volume of 500 uL
-
** 500 uLにするため450 uLのTEを加えた
+
** Centrifuged at 10000 G for more than an hour till all 4 samples volume was less then 45 uL
-
** ４サンプルすべてが45 uL以下になるまで１時間以上，10000Gで遠心した
+
* Measured the final amount of samples and added TE till all were 45 uL
-
* 遠心したサンプルの容量を量り，（元の）45 uLになるようにTEでメスアップした
+
** Used 500 uL tubes
-
** 後でLigation反応をするウォーターバスが500 uLチューブ用なので，これを使用した
+
 
 +
===Ligation System===
 +
 
 +
From PCR products we only used No.3 （[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）178 bp made on previous day
-
===ligation反応系===
 
-
PCR productsは前日に作成した３番（1-23L（terminator）178 bp）を１つだけ使用した
 
{| style="text-align:center;" class="protocol"
{| style="text-align:center;" class="protocol"
|-
|-
-
|style="border-bottom:1px solid #000; border-right:1px solid #000;"|  
+
|style="border-bottom:1px solid #996; border-right:1px solid #996;"|'''Tube No.'''
-
|style="border-bottom:1px solid #000;"|  １
+
|style="border-bottom:1px solid #996;"|  １
-
|style="border-bottom:1px solid #000;"|  ２
+
|style="border-bottom:1px solid #996;"|  ２
-
|style="border-bottom:1px solid #000;"|  ３
+
|style="border-bottom:1px solid #996;"|  ３
-
|style="border-bottom:1px solid #000;"|  ４
+
|style="border-bottom:1px solid #996;"|  ４
|-
|-
-
|style="border-right:1px solid #000;"| PCR products
+
|style="border-right:1px solid #996;"| PCR products
| 1 uL
| 1 uL
| 1 uL
| 1 uL
Line 38: Line 39:
| 1 uL
| 1 uL
|-
|-
-
|style="border-right:1px solid #000;"| 10x H Buffer
+
|style="border-right:1px solid #996;"| 10x H Buffer
| -
| -
| 1 uL
| 1 uL
Line 44: Line 45:
| 1 uL
| 1 uL
|-
|-
-
|style="border-right:1px solid #000;"| DW
+
|style="border-right:1px solid #996;"| DW
| 9 uL
| 9 uL
| 7.5 uL
| 7.5 uL
Line 50: Line 51:
| 7.5 uL
| 7.5 uL
|-
|-
-
|style="border-right:1px solid #000;"| Xba1
+
|style="border-right:1px solid #996;"| Xba1
| -
| -
| 0.5 uL
| 0.5 uL
Line 56: Line 57:
| -
| -
|-
|-
-
|style="border-right:1px solid #000; border-bottom:1px solid #000;"| Pst1
+
|style="border-right:1px solid #996; border-bottom:1px solid #996;"| Pst1
-
|style="border-bottom:1px solid #000;"| -
+
|style="border-bottom:1px solid #996;"| -
-
|style="border-bottom:1px solid #000;"| -
+
|style="border-bottom:1px solid #996;"| -
-
|style="border-bottom:1px solid #000;"| 0.5 uL
+
|style="border-bottom:1px solid #996;"| 0.5 uL
-
|style="border-bottom:1px solid #000;"| 0.5 uL
+
|style="border-bottom:1px solid #996;"| 0.5 uL
|-
|-
-
|style="border-right:1px solid #000;"| 制限酵素処理
+
|style="border-right:1px solid #996;"| Restriction Enzyme Digestion
-
| 4℃
+
| 4C
-
|colspan="3" style="background-color:#DDD7B0;"| @37℃ 60 min
+
|colspan="3" style="background-color:#DDD7B0;"| at 37C for 60 min
|-
|-
-
|style="border-right:1px solid #000;"| 制限酵素不活化
+
|style="border-right:1px solid #996;"| Restriction enzyme Inactivation
-
| colspan="4" style="background-color:#DDD7B0;"| @60℃ 15 min
+
| colspan="4" style="background-color:#DDD7B0;"| at 60C for 15 min
|-
|-
-
|style="border-right:1px solid #000;"| ligation solution
+
|style="border-right:1px solid #996;"| ligation solution
| 10 uL
| 10 uL
| 10 uL
| 10 uL
Line 75: Line 76:
| 10 uL
| 10 uL
|-
|-
-
|style="border-right:1px solid #000;"| Ligation反応
+
|style="border-right:1px solid #996;"| Ligation
-
| colspan="4" style="background-color:#DDD7B0;"|@16℃ 30 min
+
| colspan="4" style="background-color:#DDD7B0;"|at 16C for 30 min
|-
|-
-
|style="border-right:1px solid #000;"| 6x SB
+
|style="border-right:1px solid #996;"| 6x SB
| 4 uL
| 4 uL
| 4 uL
| 4 uL
Line 86: Line 87:
|}
|}
-
→1% Agarose Gel Electrophoresis in 1/2 TBE
+
→1% Agarose Gel Electrophoresis in 1/2 TBE + EtOH
-
==電気泳動==
+
 
-
* 制限酵素処理したサンプル１～４を電気泳動した
+
==Electrophoresis==
-
* マーカーはpUC119/''Hin''f
+
* Performed electrophoresis for every digested sample, 1 through 4
-
* DNA solutionが薄すぎたため，バンドが見えなかった
+
* Used marker [https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png pUC119/''Hin''f]
 +
* DNA solution was too　diluted and no bands were visible

Latest revision as of 07:27, 27 October 2010

Notebook

LB Culture

  • For every 2 mL of LB added 2 uL of antibiotics
  • Transfered a colony to LB
  • One colony didn't grow well so we isolated another one
  • Prepared more tubes for mini preps int he future

glycerol Stock

  • Added 1 mL of 80% Glycerol to screw cap tube
  • Added 1 mL of cell and broth solution to Glycerol after 2h of incubation
  • Sored at -80℃

Ligation

DNA Preparation for Ligation

  • Used 49 uL of yesterdays PCR product
  • Removed primers via Microcon YM-10
    • Added 450 uL of TE to make final volume of 500 uL
    • Centrifuged at 10000 G for more than an hour till all 4 samples volume was less then 45 uL
  • Measured the final amount of samples and added TE till all were 45 uL
    • Used 500 uL tubes

Ligation System

From PCR products we only used No.3 （1-23L（terminator）178 bp made on previous day

Tube No. １ ２ ３ ４
PCR products 1 uL 1 uL 1 uL 1 uL
10x H Buffer - 1 uL 1 uL 1 uL
DW 9 uL 7.5 uL 7.0 uL 7.5 uL
Xba1 - 0.5 uL 0.5 uL -
Pst1 - - 0.5 uL 0.5 uL
Restriction Enzyme Digestion 4C at 37C for 60 min
Restriction enzyme Inactivation at 60C for 15 min
ligation solution 10 uL 10 uL 10 uL 10 uL
Ligation at 16C for 30 min
6x SB 4 uL 4 uL 4 uL 4 uL

→1% Agarose Gel Electrophoresis in 1/2 TBE + EtOH

Electrophoresis

  • Performed electrophoresis for every digested sample, 1 through 4
  • Used marker pUC119/Hinf
  • DNA solution was too　diluted and no bands were visible
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August11"