Team:UC Chile/Cyano/Labbook/week22

From 2012.igem.org

(Difference between revisions)
(Created page with "{{UC_Chile}}")
Line 1: Line 1:
{{UC_Chile}}
{{UC_Chile}}
 +
 +
*Batch full of competent cells. OD: 0.495
 +
 +
*Ligation
 +
 +
LuxCD +LuxEG in pSB4K5 (L1) and pSB1K3 (L2).
 +
 +
*Gibson: sfGFP in pSB4K5. Assembly made to test new competent cells 
 +
 +
*Transformation
 +
 +
Plates
 +
 +
L1: Ligated LuxCD +LuxEG in pSB4K5 at 1:1 and 1:10
 +
L2: Ligated LuxCD +LuxEG in pSB1K3 at 1:1 and 1:10
 +
G+: Gibson sfGFP in pSB4K5
 +
PUC19 (AMP) x3: to test our new competent cells, 20µL of transformed bacteria were plated.
 +
 +
Tuesday
 +
 +
*Transformation results
 +
 +
PUC19-1: 148 colonies
 +
PUC19-2: 177 colonies
 +
PUC19-3: 155 colonies
 +
L1 and L2 with colonies. Colony PCR will be done.
 +
G+ Positive results. Several colonies. Probably due to: the problem with the Gibson assembly could be the use of E. cloni instead of TOP10.
 +
 +
*Colony PCR for LuxCDEG in pSB1K3: Negative. PCR will be tried.
 +
*Colony PCR for LuxCDEG in pSB4K5 was not done due to all colonies that grew were red. It was assumed LUXCDEG did not integrate.
 +
*PCR for RS1-Kan-B0015-RS2 (chloramphenicol resistance) and electrophoresis: primers form dimers. PCR will be repeated at higher Tm.
 +
 +
*PCR run:
 +
 +
 +
Name PCR           Template   Primer F Primer R Length
 +
 
 +
1 RS1 KAN B0015 RS2                  F8    F5         4300
 +
 
 +
2 KAN B0015 RS2                      F8         F2         3760
 +
 +
Notes
 +
 +
*It was a touch-Down PCR
 +
1 and 2 amplified and had right size. Only 1 will be purified because is more useful than 2.
 +
 +
 +
Wednesday
 +
 +
* Band purification
 +
 +
Name              PCR date        Concentration ng/µl
 +
 +
C4-Open          07.31            21,2
 +
Pcaa3 promoter    07.27            18,1
 +
 +
* PCR run:
 +
 +
 +
Name PCR         Template     Primer F       Primer R         Length  
 +
1 SigEP-LuxCDEG K325005             16.H1       16.H4         4000  
 +
2 Pcaa3-LuxCDEG K325005             16.H9       16.H4         4000  
 +
3 SigEP 4 LuxCDEG Cyano             16.G9       16.H2  
 +
4 Pcaa3 4 LuxCDEG Cyano             16.H10       16.H7         101-141  
 +
5 taP (BB) Cyano             16.I1       16.I4         150-190  
 +
6 taP_VB  pSB1C3             16.I2       16.I3         2000  
 +
7 Pcaa3 (BB) Cyano             16.I5       16.I8         101-141  
 +
8 Pcaa3_VB pSB1C3             16.I6       16.I7         2000  
 +
9 taP-LuxAB Cyano             16.G3       16.G6         150-190  
 +
10 LuxAB in CK K325009              16.G5       16.G8
 +
 +
* Gibson assembly:
 +
 +
C1.1: C4-Open+pSBAB+mRFP  (CK)
 +
C1.2: C4-Open+pSBA2+mRFP  (CK)
 +
 +
Thursday
 +
 +
*Band purification
 +
 +
 +
Name PCR                   Concentration [ng/µl]
 +
 
 +
1 <b>SigEP</b>-LuxCDEG   26.1  
 +
2 <b>Pcaa3 4</b>LuxCDEG   8.8  
 +
3 taP_<b>VB</b>           12.6  
 +
4 Pcaa3_<b>VB</b>          20.3  
 +
5 taP-<b>LuxAB</b>   26.6  
 +
6 ADF-3+                   13.0  
 +
7 Pcaa3 4 BB           13.8
 +
 +
Notes: amplicons in bold

Revision as of 21:27, 9 August 2012

Cyanolux & SpiderColi - Pontificia Universidad Católica de Chile, iGEM 2012



  • Batch full of competent cells. OD: 0.495
  • Ligation

LuxCD +LuxEG in pSB4K5 (L1) and pSB1K3 (L2).

  • Gibson: sfGFP in pSB4K5. Assembly made to test new competent cells
  • Transformation

Plates

L1: Ligated LuxCD +LuxEG in pSB4K5 at 1:1 and 1:10 L2: Ligated LuxCD +LuxEG in pSB1K3 at 1:1 and 1:10 G+: Gibson sfGFP in pSB4K5 PUC19 (AMP) x3: to test our new competent cells, 20µL of transformed bacteria were plated.

Tuesday

  • Transformation results

PUC19-1: 148 colonies PUC19-2: 177 colonies PUC19-3: 155 colonies L1 and L2 with colonies. Colony PCR will be done. G+ Positive results. Several colonies. Probably due to: the problem with the Gibson assembly could be the use of E. cloni instead of TOP10.

  • Colony PCR for LuxCDEG in pSB1K3: Negative. PCR will be tried.
  • Colony PCR for LuxCDEG in pSB4K5 was not done due to all colonies that grew were red. It was assumed LUXCDEG did not integrate.
  • PCR for RS1-Kan-B0015-RS2 (chloramphenicol resistance) and electrophoresis: primers form dimers. PCR will be repeated at higher Tm.
  • PCR run:


Name PCR Template Primer F Primer R Length

1 RS1 KAN B0015 RS2 F8 F5 4300

2 KAN B0015 RS2 F8 F2 3760

Notes

  • It was a touch-Down PCR

1 and 2 amplified and had right size. Only 1 will be purified because is more useful than 2.


Wednesday

  • Band purification

Name PCR date Concentration ng/µl

C4-Open 07.31 21,2 Pcaa3 promoter 07.27 18,1

  • PCR run:


Name PCR Template Primer F Primer R Length 1 SigEP-LuxCDEG K325005 16.H1 16.H4 4000 2 Pcaa3-LuxCDEG K325005 16.H9 16.H4 4000 3 SigEP 4 LuxCDEG Cyano 16.G9 16.H2 4 Pcaa3 4 LuxCDEG Cyano 16.H10 16.H7 101-141 5 taP (BB) Cyano 16.I1 16.I4 150-190 6 taP_VB pSB1C3 16.I2 16.I3 2000 7 Pcaa3 (BB) Cyano 16.I5 16.I8 101-141 8 Pcaa3_VB pSB1C3 16.I6 16.I7 2000 9 taP-LuxAB Cyano 16.G3 16.G6 150-190 10 LuxAB in CK K325009 16.G5 16.G8

  • Gibson assembly:

C1.1: C4-Open+pSBAB+mRFP (CK) C1.2: C4-Open+pSBA2+mRFP (CK)

Thursday

  • Band purification


Name PCR Concentration [ng/µl]

1 SigEP-LuxCDEG 26.1 2 Pcaa3 4LuxCDEG 8.8 3 taP_VB 12.6 4 Pcaa3_VB 20.3 5 taP-LuxAB 26.6 6 ADF-3+ 13.0 7 Pcaa3 4 BB 13.8

Notes: amplicons in bold

Retrieved from "http://2012.igem.org/Team:UC_Chile/Cyano/Labbook/week22"