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Team:UC Chile/Cyano/Labbook/week22
From 2012.igem.org
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* Bands 6 and 8 were really faint but had the right size. | * Bands 6 and 8 were really faint but had the right size. | ||
* Amplicons in bold | * Amplicons in bold | ||
| + | |||
| + | Friday | ||
| + | |||
| + | *Band purification | ||
| + | |||
| + | |||
| + | Name PCR Concentration [ng/µl] | ||
| + | |||
| + | 1 pBAD-<b>ADF-3</b> 9.7 | ||
| + | 6 <b>LuxCDEG</b> (sigEP) 5.6 | ||
| + | 8 <b>LuxCDEG</b> (pcaa3) 4.1 | ||
| + | 10 pcaa3 LuxCDEG - <b>VB IntC</b> 14.8 | ||
| + | 11 psigEP LuxCDEG – <b>VB IntC</b> 33.2 | ||
| + | 13 <b>LuxAB</b> (Xenor..lumins..) 6.1 | ||
| + | |||
| + | Notes | ||
| + | Parts 6 and 8 (LuxCDEG) will be used for a new PCR because they were very faint. | ||
| + | |||
| + | *Gibson Isothermal Buffer | ||
| + | |||
| + | A new buffer was made because we realized the old one was prepared with wrong concentrations. New concentrations: MgCL2 2M, DTT 1M. | ||
| + | |||
| + | *Gibson and transformation | ||
| + | |||
| + | C1.1: C4-Open+pSBAB+mRFP (CK) | ||
| + | C1.2: C4-Open+pSBA2+mRFP (CK) | ||
| + | Pcaa3 BB: Pcaa3 BB + VB_Pcaa3 (C) | ||
| + | ADF 3: ADF3 + (pBAD) VB_ADF3 (C) | ||
| + | *Error: mRFP was used for C1.2 instead of RFP (C1.2 is designed to work with RFP). | ||
| + | *C4 Open: RS1+KAN+B0015+RS2 | ||
| + | |||
| + | *Colony PCR | ||
| + | |||
| + | C1.1 and C1.2 (assembly date 08.01): 20 colonies were selected from each plate. Right sized colonies: 5,6,7,8 and eleven from C1.1 and 11 from C1.2. Maybe colony 27 from C1.2. This Gibson assembly was repeated anyway. Colony 6 did not grow. | ||
Latest revision as of 21:47, 9 August 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
- Gibson results: plate agar was dry. Colony PCR will be done of surviving colonies.
- PCR Run
Name PCR Template Primer F Primer R Length
1 pBAD-ADF-3 ADF-3 16.F9 F8 2000
2 pBAD-ADF-3 ADF-3 16.F9 F8 2000
3 ADF-3+ VB LuxBrick 16.F7 F10 3000
4 ADF-3+ VB LuxBrick 16.Fn F10 3000
5 tap-BB Cyano DNA 16.I1 I4 140-190
6 LuxCDEG (sigEP) K3259015 16.H1 H4 4000
7 LuxCDEG (sigEP) K3259005 16.H1 H4
8 LuxCDEG (pcaa3) K3259015 16.H9 H4 4000
9 LuxCDEG (pcaa3) K3259005 16.H9 H4
10 pcaa3 LuxCDEG - VB IntC psbA2-IntC 16.H3 16.H8 4000
11 psigEP LuxCDEG – VB IntC psbA2-IntC 16.H3 16.G10 4000
12 LuxAB K328905 16.G5 16.G8 2000
13 LuxAB K352905 16.G5 16.G8 2000
14 LuxAB - VB ->Need C K
Notes
- Bands 6 and 8 were really faint but had the right size.
- Amplicons in bold
Friday
- Band purification
Name PCR Concentration [ng/µl]
1 pBAD-ADF-3 9.7 6 LuxCDEG (sigEP) 5.6 8 LuxCDEG (pcaa3) 4.1 10 pcaa3 LuxCDEG - VB IntC 14.8 11 psigEP LuxCDEG – VB IntC 33.2 13 LuxAB (Xenor..lumins..) 6.1
Notes Parts 6 and 8 (LuxCDEG) will be used for a new PCR because they were very faint.
- Gibson Isothermal Buffer
A new buffer was made because we realized the old one was prepared with wrong concentrations. New concentrations: MgCL2 2M, DTT 1M.
- Gibson and transformation
C1.1: C4-Open+pSBAB+mRFP (CK) C1.2: C4-Open+pSBA2+mRFP (CK) Pcaa3 BB: Pcaa3 BB + VB_Pcaa3 (C) ADF 3: ADF3 + (pBAD) VB_ADF3 (C)
- Error: mRFP was used for C1.2 instead of RFP (C1.2 is designed to work with RFP).
- C4 Open: RS1+KAN+B0015+RS2
- Colony PCR
C1.1 and C1.2 (assembly date 08.01): 20 colonies were selected from each plate. Right sized colonies: 5,6,7,8 and eleven from C1.1 and 11 from C1.2. Maybe colony 27 from C1.2. This Gibson assembly was repeated anyway. Colony 6 did not grow.
