Team:Groningen/Notebook/30 July 2009

From 2009.igem.org

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===Transporters===
===Transporters===
-
GlpF
+
'''GlpF'''
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+
-
Redone PCR3
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 +
:*Redone PCR3
{|
{|
|25uL
|25uL
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|}
|}
-
The resulting PCR product was purified using a PCR purification kit of Roche.
+
The resulting PCR product was purified using a high pure PCR product purification kit (Roche).
concentration was measured using Nanodrop;
concentration was measured using Nanodrop;
{|
{|
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The resticted GlpF PCR3 (30/07) was ligated with psB1AC3 resticted with EcoRI and SpeI.
The resticted GlpF PCR3 (30/07) was ligated with psB1AC3 resticted with EcoRI and SpeI.
-
Ligation mix:
+
:*Ligation mix:
{|
{|
|2.0 uL
|2.0 uL
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|GlpF PCR3
|GlpF PCR3
|-
|-
-
|
+
|}
===Metal Accumulation===
===Metal Accumulation===
===Vectors===
===Vectors===
-
[http://openwetware.org/wiki/User:Wilfred_J._Poppinga/Notebook/Wilfreds_Project/2009/07/30 Results] were disappointing, no colonies on plate. Hope the fresh ligase and ligase buffer (10x) will help the new ligation that was started.
+
[http://openwetware.org/wiki/User:Wilfred_J._Poppinga/Notebook/Wilfreds_Project/2009/07/30 Results] were disappointing, no colonies on plate. Hope the fresh ligase and ligase buffer (10x) will help the new ligation that was started. New vectors were cut and purified from gel. <BR><center>[[Image:PSB1AC3_%26_pSB3K3_cut_with_SpeI_%26_EcoRI.jpg|500xpx]]</center>
 +
 
 +
*'''Ligation (ratio 1:3) of pSB1AC3-HML and pSB3K3-HML + RFP and pSB3K3 and pSB1AC3 + pLac'''
 +
**Calculated the volumes of vector and insert with Cloning Tools 6.0, using:
 +
***Amount: 200ng
 +
***Excess insert: 3x
 +
In a total volume of 20uL, 2uL 10x Ligase Buffer and 1uL T4 DNA Ligase.
 +
**The fragments were ligated using PCR program AC3 ligation:
 +
{|Border=1
 +
! AC3 ligation program
 +
! Temperature
 +
! Time
 +
|-
 +
|DNA Denaturing
 +
|42°
 +
|2 min
 +
|-
 +
|Ligation
 +
|16°
 +
|1 hr
 +
|-
 +
|Protein inactivation
 +
|65°
 +
|10 min
 +
|}
 +
**Use 5uL ligation mixture for transformation.
 +
**Transform ''E. coli'' TOP10 via normal protocol.
==Dry==
==Dry==

Latest revision as of 16:31, 4 August 2009

Igemhomelogo.png

Wet

GVP Cluster

Transporters

GlpF

  • Redone PCR3
25uL Phusion
2 uL GlpF Fw
2 uL GlpF Rev
0.5 uL GlpF PCR3 (28/07/09) DNA
21uL MQ

The resulting PCR product was purified using a high pure PCR product purification kit (Roche). concentration was measured using Nanodrop;

16,0 ng/ul GlpFPCR3 30/07
36.4 ng/ul psB1AC3-M digested
32.0 ng/uL GlpF digested

Restiction was done on the GlpF PCR3 (30/07) with XbaI and PstI as noted on the notebook of 28/07/09 The resticted GlpF PCR3 (30/07) was ligated with psB1AC3 resticted with EcoRI and SpeI.

  • Ligation mix:
2.0 uL T4 Ligase buffer
0.5 uL T4 Ligase
11.25 uL GlpF PCR3

Metal Accumulation

Vectors

Results were disappointing, no colonies on plate. Hope the fresh ligase and ligase buffer (10x) will help the new ligation that was started. New vectors were cut and purified from gel.
PSB1AC3 & pSB3K3 cut with SpeI & EcoRI.jpg
  • Ligation (ratio 1:3) of pSB1AC3-HML and pSB3K3-HML + RFP and pSB3K3 and pSB1AC3 + pLac
    • Calculated the volumes of vector and insert with Cloning Tools 6.0, using:
      • Amount: 200ng
      • Excess insert: 3x

In a total volume of 20uL, 2uL 10x Ligase Buffer and 1uL T4 DNA Ligase.

    • The fragments were ligated using PCR program AC3 ligation:
AC3 ligation program Temperature Time
DNA Denaturing 42° 2 min
Ligation 16° 1 hr
Protein inactivation 65° 10 min
    • Use 5uL ligation mixture for transformation.
    • Transform E. coli TOP10 via normal protocol.

Dry

We worked on migrating graphs from Google spreadsheets to Wiki tables, making sure that at least the raw data used to generate the graphs is "safe".


April
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July
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October
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