Team:HokkaidoU Japan/Notebook/August11

From 2010.igem.org

(Difference between revisions)
(→Ligation System)
(→Ligation System)
Line 27: Line 27:
{| 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℃
| 4℃
|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
Line 76: Line 76:
| 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

Revision as of 06:53, 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

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 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

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"