Team:HokkaidoU Japan/Notebook/August11

From 2010.igem.org

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Line 17: Line 17:
* Removed primers via Microcon YM-10
* Removed primers via Microcon YM-10
** Added 450 uL of TE to make final volume of 500 uL
** 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
+
** 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
* Measured the final amount of samples and added TE till all were 45 uL
** Used 500 uL tubes
** Used 500 uL tubes
Line 23: Line 23:
===Ligation System===
===Ligation System===
-
From PCR products we only used No.3 （1-23L（terminator）178 bp made on previous day
+
From PCR products we only used No.3 （[[Team:HokkaidoU_Japan/Parts#BioBricks|1-23L]]（terminator）178 bp made on previous day
{| style="text-align:center;" class="protocol"
{| style="text-align:center;" class="protocol"
|-
|-
-
|style="border-bottom:1px solid #000; border-right:1px solid #000;"|'''Tube No.'''
+
|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 39: 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 45: 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 51: 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 57: 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;"| Restriction Enzyme Digestion
+
|style="border-right:1px solid #996;"| Restriction Enzyme Digestion
-
| 4℃
+
| 4C
|colspan="3" style="background-color:#DDD7B0;"| at 37C for 60 min
|colspan="3" style="background-color:#DDD7B0;"| at 37C for 60 min
|-
|-
-
|style="border-right:1px solid #000;"| Restriction enzyme Inactivation
+
|style="border-right:1px solid #996;"| Restriction enzyme Inactivation
| colspan="4" style="background-color:#DDD7B0;"| at 60C for 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
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| 10 uL
| 10 uL
|-
|-
-
|style="border-right:1px solid #000;"| Ligation
+
|style="border-right:1px solid #996;"| Ligation
| colspan="4" style="background-color:#DDD7B0;"|at 16C for 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 87: Line 87:
|}
|}
-
→1% Agarose Gel Electrophoresis in 1/2 TBE + EtOh
+
→1% Agarose Gel Electrophoresis in 1/2 TBE + EtOH
==Electrophoresis==
==Electrophoresis==

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
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