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Team:UC Chile/Cyano/Labbook/week23
From 2012.igem.org
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* PCR run | * PCR run | ||
| - | + | <table class="wikitable"> | |
| - | 1 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 8%Dmso | + | <tr> |
| - | 2 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 5%Dmso | + | <td> |
| - | 3 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 5%Dmso | + | </td><td>pBAD |
| - | 4 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 8%Dmso | + | </td><td>PP Luc (1) |
| - | 5 LuxBrick nP LuxBrick 17.E1 Suffix.Dig R 600 | + | </td><td>PP Luc (2) |
| - | 6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000 | + | </td><td>LC Luc (1) |
| - | 7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000 | + | </td></tr> |
| - | 8 ADF-3 Alone ADF-3 17.E2 17.E3 2000 | + | <tr> |
| - | 9 BBricking VB (V.fis) PSB1C3 (RS1) Suffix.Dig F Preffix.Dig R 2000 | + | <td>Nuclease-free H20 |
| - | 10 BBricking VB (V.fis) PSB1T3 Suffix.Dig F Preffix.Dig R 2000 | + | </td><td>14 |
| - | 11 ADF-3_VB LuxBrick 16.F7 16.F10 3000 | + | </td><td>14 |
| - | 12 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000 | + | </td><td>14 |
| - | 13 ADF-3_VB LuxBrick 16.F7 16.F10 3000 | + | </td><td>14 |
| - | 14 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000 | + | </td></tr> |
| + | <tr> | ||
| + | <td>10x FD Buffer | ||
| + | </td><td>2 | ||
| + | </td><td>2 | ||
| + | </td><td>2 | ||
| + | </td><td>2 | ||
| + | </td></tr> | ||
| + | <tr> | ||
| + | <td>Plasmid DNA | ||
| + | </td><td>2 | ||
| + | </td><td>2 | ||
| + | </td><td>2 | ||
| + | </td><td>2 | ||
| + | </td></tr> | ||
| + | <tr> | ||
| + | <td>FD EcoRI | ||
| + | </td><td>1 | ||
| + | </td><td>1 | ||
| + | </td><td>1 | ||
| + | </td><td>1 | ||
| + | </td></tr> | ||
| + | <tr> | ||
| + | <td>FD SpeI | ||
| + | </td><td>1 | ||
| + | </td><td>0 | ||
| + | </td><td>0 | ||
| + | </td><td>0 | ||
| + | </td></tr> | ||
| + | <tr> | ||
| + | <td>FD XbaI | ||
| + | </td><td>0 | ||
| + | </td><td>1 | ||
| + | </td><td>1 | ||
| + | </td><td>1 | ||
| + | </td></tr></table> | ||
| + | |||
| + | |||
| + | Name PCR Template Primer F Primer R Length Additive<br><br> | ||
| + | |||
| + | 1 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 8%Dmso<br> | ||
| + | 2 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 5%Dmso<br> | ||
| + | 3 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 5%Dmso<br> | ||
| + | 4 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 8%Dmso<br> | ||
| + | 5 LuxBrick nP LuxBrick 17.E1 Suffix.Dig R 600<br> | ||
| + | 6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000<br> | ||
| + | 7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000<br> | ||
| + | 8 ADF-3 Alone ADF-3 17.E2 17.E3 2000<br> | ||
| + | 9 BBricking VB (V.fis) PSB1C3 (RS1) Suffix.Dig F Preffix.Dig R 2000<br> | ||
| + | 10 BBricking VB (V.fis) PSB1T3 Suffix.Dig F Preffix.Dig R 2000<br> | ||
| + | 11 ADF-3_VB LuxBrick 16.F7 16.F10 3000<br> | ||
| + | 12 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000<br> | ||
| + | 13 ADF-3_VB LuxBrick 16.F7 16.F10 3000<br> | ||
| + | 14 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000<br> | ||
Notes | Notes | ||
Latest revision as of 22:56, 9 August 2012
Monday:
- PCR colony (Gibson assembly date 08.03)
- PCR run
| pBAD | PP Luc (1) | PP Luc (2) | LC Luc (1) | |
| Nuclease-free H20 | 14 | 14 | 14 | 14 |
| 10x FD Buffer | 2 | 2 | 2 | 2 |
| Plasmid DNA | 2 | 2 | 2 | 2 |
| FD EcoRI | 1 | 1 | 1 | 1 |
| FD SpeI | 1 | 0 | 0 | 0 |
| FD XbaI | 0 | 1 | 1 | 1 |
Name PCR Template Primer F Primer R Length Additive
1 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 8%Dmso
2 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 5%Dmso
3 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 5%Dmso
4 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 8%Dmso
5 LuxBrick nP LuxBrick 17.E1 Suffix.Dig R 600
6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000
7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000
8 ADF-3 Alone ADF-3 17.E2 17.E3 2000
9 BBricking VB (V.fis) PSB1C3 (RS1) Suffix.Dig F Preffix.Dig R 2000
10 BBricking VB (V.fis) PSB1T3 Suffix.Dig F Preffix.Dig R 2000
11 ADF-3_VB LuxBrick 16.F7 16.F10 3000
12 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000
13 ADF-3_VB LuxBrick 16.F7 16.F10 3000
14 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000
Notes
- PCR Tm=56°C.
- 11 and 12 Tm=60°C
- 13 and 14 TouchDown PCR. PCR annealing program 54°C.
Tuesday:
- PCR band recuperation
Name PCR Concentration [ng/µl] 1 and 2 psigEP -LuxCDEG 5.7
- Digestion of colonies selected in colony PCR constructs C1.1 and C1.2 (Gibson assembly date: 08.01)
C1.1: Band from colony 8 is the right size. Construct succesfully assembled and will be sequenced to confirm. C1.2 : Wrong size (all colonies). Probably due to: the use of mRFP instead of RFP for the Gibson assembly.
- Gibson results (assembly date: 08.03) and Colony PCR
C1.1: two positive colonies but were the wrong size. C1.2: several positive colonies but were the wrong size. Probably due to: the use of mRFP instead of RFP for the Gibson assembly. Pcaa3 BB: several colonies and with the right size. It will be digested. ADF 3: no colonies.
- Miniprep and positive colonies digestion (Gibson assembly date: 08.03)
Colony Concentration [ng/µl] 1 52.4 2 45.8 5 38.3 10 62.9
- Synechocystis PCC6803 transformation results (transformation date: 07.20)
Synechocystis did not grow. Probably due to: they die in the presence of glucose. Synechocystis were reinoculated and e. coli was transformed with psbAB+GFP to further transformation in synechocystis.
- PCR run (59ºC)
Name PCR Template Primer F Primer R Length Additive
1 Bactomithril
2 Bactomithril
4 Pcaa3 -LuxCDEG sigEP -LuxCDEG 16.H9 16.H4 4000 8%Dmso
5 LuxBrick (No P) LuxBrick 17.E1 Suffix.Dig R 600 5%Dmso
6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000 5%Dmso
7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000 5%Dmso
8 ADF-3 Alone ADF-3 17.E2 17.E3 2000 5%Dmso
9 C1_R_VB C1.1 16.E7 16.E6 4300
10 C1.2_VB C1.1 14.F6 14.F5 5200
Notes
- 9: amplifies from RS2 to mRFP. Switches Kan to KanR.
- 10: amplifies from mRFP to RS1. Switches psbAB to psbAB2 (C1.2)
