Team:HokkaidoU Japan/Notebook/August11

From 2010.igem.org

(Difference between revisions)
(→ligation用DNAの調製)
(→ligation反応系)
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** Used 500 uL tubes
** Used 500 uL tubes
-
===ligation反応系===
+
===Ligation System===
-
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
+
 
 +
From PCR products we only used No.3 （1-23L（terminator）178 bp made on previous day
 +
 
{| style="text-align:center;" class="protocol"
{| style="text-align:center;" class="protocol"
|-
|-
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|style="border-bottom:1px solid #000;"| 0.5 uL
|style="border-bottom:1px solid #000;"| 0.5 uL
|-
|-
-
|style="border-right:1px solid #000;"| 制限酵素処理
+
|style="border-right:1px solid #000;"| Restriction Enzyme Digestion
| 4℃
| 4℃
-
|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 #000;"| 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 #000;"| ligation solution
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| 10 uL
| 10 uL
|-
|-
-
|style="border-right:1px solid #000;"| Ligation反応
+
|style="border-right:1px solid #000;"| 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 #000;"| 6x SB
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|}
|}
-
→1% Agarose Gel Electrophoresis in 1/2 TBE
+
→1% Agarose Gel Electrophoresis in 1/2 TBE + EtOh
 +
 
==電気泳動==
==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
* 制限酵素処理したサンプル１～４を電気泳動した
* マーカーはpUC119/''Hin''f
* マーカーはpUC119/''Hin''f
* DNA solutionが薄すぎたため，バンドが見えなかった
* DNA solutionが薄すぎたため，バンドが見えなかった

Revision as of 06:22, 20 September 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 10000G 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

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 4℃ 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

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
  • マーカーはpUC119/Hinf
  • DNA solutionが薄すぎたため，バンドが見えなかった
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