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Team:USP-UNESP-Brazil/Notebook
From 2012.igem.org
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====MR3==== | ====MR3==== | ||
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09/07/12 - Amanda, Cleandho, Débora and Lucas: | 09/07/12 - Amanda, Cleandho, Débora and Lucas: | ||
- Bacterial transformation with MR3 part. | - Bacterial transformation with MR3 part. | ||
MR3 – grew colonies | MR3 – grew colonies | ||
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10/07/2012 - Amanda, Cleandho, Débora and Lucas: | 10/07/2012 - Amanda, Cleandho, Débora and Lucas: | ||
- Bacterial clones from MR3 were inoculated in LB with tetracycline | - Bacterial clones from MR3 were inoculated in LB with tetracycline | ||
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11/07/2012 – Amanda, Cleandho, Débora and Lucas: | 11/07/2012 – Amanda, Cleandho, Débora and Lucas: | ||
- Plasmid extraction of MR3 | - Plasmid extraction of MR3 | ||
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- Digestion with enzymes and incubate 37 ° C for 1 hour | - Digestion with enzymes and incubate 37 ° C for 1 hour | ||
MR3 – ( EcoRI and SpeI) | MR3 – ( EcoRI and SpeI) | ||
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11/07/2012 – Aline and Cleandho: | 11/07/2012 – Aline and Cleandho: | ||
- Electrophoresis to confirmation: no DNA yield | - Electrophoresis to confirmation: no DNA yield | ||
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18/07/12 – Aline and Cleandho: | 18/07/12 – Aline and Cleandho: | ||
- Ligation of the part MR3 using pSB1C3 backbone | - Ligation of the part MR3 using pSB1C3 backbone | ||
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19.07.12 – Aline, Cleandho and Débora: | 19.07.12 – Aline, Cleandho and Débora: | ||
- Transformation of MR3. | - Transformation of MR3. | ||
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23/07/12 – Cleandho and Débora: | 23/07/12 – Cleandho and Débora: | ||
- Quantification of pDNA: | - Quantification of pDNA: | ||
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- Digestion, using approximately 300ng of DNA | - Digestion, using approximately 300ng of DNA | ||
MR3: EcoRI + SpeI | MR3: EcoRI + SpeI | ||
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24/07/12 – Cleandho: | 24/07/12 – Cleandho: | ||
- Electrophoresis to confirmation of MR3-1 clone: no DNA yield | - Electrophoresis to confirmation of MR3-1 clone: no DNA yield | ||
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07/08/12 - Cleandho and Lucas: | 07/08/12 - Cleandho and Lucas: | ||
- Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3 | - Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3 | ||
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09/08/12 – Amanda | 09/08/12 – Amanda | ||
- Transformation of MR3 assembly | - Transformation of MR3 assembly | ||
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14/08/12 – Lucas and Amanda | 14/08/12 – Lucas and Amanda | ||
- Plasmid extraction from two MR3 clones | - Plasmid extraction from two MR3 clones | ||
- Digestion of MR3 clones with (EcoRI + SpeI) | - Digestion of MR3 clones with (EcoRI + SpeI) | ||
- Electrophoresis to confirm MR3 clones: result failed | - Electrophoresis to confirm MR3 clones: result failed | ||
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22/08 – Cleandho and Lucas: | 22/08 – Cleandho and Lucas: | ||
- Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) | - Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) | ||
- Transformation of MR3 assembly | - Transformation of MR3 assembly | ||
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04/09/12 – Cleandho: | 04/09/12 – Cleandho: | ||
- Plasmid extraction of six MR3 clones | - Plasmid extraction of six MR3 clones | ||
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- Digestion of MR3 using HindIII | - Digestion of MR3 using HindIII | ||
- Electrophoresis of MR3 digested with HindIII. Result: all clones have failed | - Electrophoresis of MR3 digested with HindIII. Result: all clones have failed | ||
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05/09/12 – Cleandho and Débora: | 05/09/12 – Cleandho and Débora: | ||
- Digestion of pSB1K3, pSB1C3, 2M, 4G | - Digestion of pSB1K3, pSB1C3, 2M, 4G | ||
- Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3 | - Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3 | ||
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06/09/12 – Cleandho: | 06/09/12 – Cleandho: | ||
- Transformation of MR3 assembly in three molar proportions. | - Transformation of MR3 assembly in three molar proportions. | ||
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11/09/12 – Cleandho and Lucas: | 11/09/12 – Cleandho and Lucas: | ||
- PCR screening of ten colonies clones of assembly MR3 | - PCR screening of ten colonies clones of assembly MR3 | ||
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12/09/12 – Cleandho and Lucas: | 12/09/12 – Cleandho and Lucas: | ||
- Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon | - Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon | ||
- Inoculum of clone 4 of MR3 in LBK | - Inoculum of clone 4 of MR3 in LBK | ||
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13/09/12 Cleandho and Lucas: | 13/09/12 Cleandho and Lucas: | ||
- Plasmid extraction of clone 4 from MR3 assembly | - Plasmid extraction of clone 4 from MR3 assembly | ||
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====MR6==== | ====MR6==== | ||
Revision as of 13:40, 17 September 2012
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Greg's lab diary (separated by assembly)
Contents |
GENERAL EXPERIMENTS
17/07/12 – Amanda and Cleandho: - Tetracicline test using TOP10 without plasmid: successful, antibiotic is working
18/07/12 – Aline and Cleandho: - Inoculum of pSB1C3, pSB1K3 and pSB1A2 using RFP as reporter
02/08/12 – Cleandho and Lucas: - Plasmid extraction of pSB1A2, pSB1K3 and pSB1C3 for storage and use. - Make competent cells for storage and use in -80°C
03/08/12 – Cleandho and Lucas: - Competent cells test for viability and competency
22/08 – Cleandho and Lucas: - Make competent cell for use and storage at -80°C - Viability and competency tests of competent TOP 10
23/08/12 – Débora: - Digestion of pSB1K3 with PstI and EcoRI
03/09/12 – Cleandho: - Preparation of LB culture media
05/09/12 – Cleandho and Débora:
- Digestion of pSB1K3, pSB1C3
MR2
09/07/12 - Amanda, Cleandho, Débora and Lucas: - Bacterial transformation with MR2 part. MR2 grew colonies
10/07/2012 - Amanda, Cleandho, Débora and Lucas: - Bacterial clones from MR2 were inoculated in LB with tetracycline
11/07/2012 – Amanda, Cleandho, Débora and Lucas: - Plasmid extraction of MR2 and quantification by nanodrop Expected size: MR2 – 25 ng/ul 11J: 82 pb + 12M: 876 pb = 958 pb - Digestion with enzymes and incubate 37 ° C for 1 hour MR2 – (XbaI and PstI)
11/07/2012 – Aline and Cleandho: - Electrophoresis to confirmation: no DNA yield
17/07/12 – Amanda and Cleandho: - Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell 18/07/12 – Aline and Cleandho: - Ligation of the parts MR2 using pSB1C3 backbone
19.07.12 – Aline, Cleandho and Débora: - Transformation of MR2
23/07/12 – Cleandho and Débora: - Quantification of pDNA: MR2-2: 119,9 ng/uL - Digestion, using approximately 300ng of DNA MR2: XbaI + PstI
24/07/12 – Cleandho: - Electrophoresis to confirmation of MR2-1 andMR2-2 clones: MR2-2 is positive and MR2-1 was negative.
MR3
09/07/12 - Amanda, Cleandho, Débora and Lucas: - Bacterial transformation with MR3 part. MR3 – grew colonies
10/07/2012 - Amanda, Cleandho, Débora and Lucas: - Bacterial clones from MR3 were inoculated in LB with tetracycline
11/07/2012 – Amanda, Cleandho, Débora and Lucas: - Plasmid extraction of MR3 Expected size: MR3 – 28 ng/ul 2M: 12 pb + 4G: 669 pb = 681 pb - Digestion with enzymes and incubate 37 ° C for 1 hour MR3 – ( EcoRI and SpeI)
11/07/2012 – Aline and Cleandho: - Electrophoresis to confirmation: no DNA yield
18/07/12 – Aline and Cleandho: - Ligation of the part MR3 using pSB1C3 backbone
19.07.12 – Aline, Cleandho and Débora: - Transformation of MR3.
23/07/12 – Cleandho and Débora: - Quantification of pDNA: MR3-1: 110,8 ng/uL - Digestion, using approximately 300ng of DNA MR3: EcoRI + SpeI
24/07/12 – Cleandho: - Electrophoresis to confirmation of MR3-1 clone: no DNA yield
07/08/12 - Cleandho and Lucas: - Digestion of 2M with EcoRI + SpeI and 4G with SbaI + PstI for assembly to MR3
09/08/12 – Amanda - Transformation of MR3 assembly
14/08/12 – Lucas and Amanda - Plasmid extraction from two MR3 clones - Digestion of MR3 clones with (EcoRI + SpeI) - Electrophoresis to confirm MR3 clones: result failed
22/08 – Cleandho and Lucas: - Ligation of assembly MR3 using pSB1K3 (this step was exclusively incubated for 2 hours, all the other ligation steps were incubated overnight) - Transformation of MR3 assembly
04/09/12 – Cleandho: - Plasmid extraction of six MR3 clones - Digestion of MR3 clones with EcoRI and SpeI - Quantification of DNA of MR3 clones: A 178 ng/uL B 129 ng/uL C 119 ng/uL D 98 ng/uL E 159 ng/uL F 110 ng/uL - Digestion of MR3 using HindIII - Electrophoresis of MR3 digested with HindIII. Result: all clones have failed
05/09/12 – Cleandho and Débora: - Digestion of pSB1K3, pSB1C3, 2M, 4G - Ligation of MR3 using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3
06/09/12 – Cleandho: - Transformation of MR3 assembly in three molar proportions.
11/09/12 – Cleandho and Lucas: - PCR screening of ten colonies clones of assembly MR3
12/09/12 – Cleandho and Lucas: - Electrophoresis of PCR screening clones of MR3 assembly. Result: MR3 shown the expected size of amplicon - Inoculum of clone 4 of MR3 in LBK
13/09/12 Cleandho and Lucas: - Plasmid extraction of clone 4 from MR3 assembly - Digestion of pDNA of clones 4 using EcoRI and SpeI - Electrophoresis for confirmation of clone 4. Result: failed
MR6
09/07/12 - Amanda, Cleandho, Débora and Lucas: - Bacterial transformation MR6 part. MR6 – not grown colonies 17/07/12 – Amanda and Cleandho: - Plasmid extraction using BIRNBOIM & DOLY (1979) protocol: failed due non lysis of the cell 18/07/12 – Aline and Cleandho: - Ligation of the part MR6 using pSB1C3 backbone 19.07.12 – Aline, Cleandho and Débora: - Transformation of MR6 23/07/12 – Cleandho and Débora: - Quantification of pDNA: MR6-1: 45,6ng/uL MR6-5: 46,7ng/uL - Digestion, using approximately 300ng of DNA MR6: EcroRI + SpeI 24/07/12 – Cleandho: - Electrophoresis to confirmation of MR6-1 and MR6-5 clones: all clones have failed 02/08/12 – Cleandho and Lucas: - Ligation/Assembly 3A of MR6 part 03/08/12 – Cleandho and Lucas: - Transformation of MR6 08/08/12 – Débora: - Plasmid extraction from MR6 clones - Digestion of pDNA with EcoRI + PstI from MR6 clones 09/08/12 – Amanda - Electrophoresis to confirm MR6 clones. Result: failed 03/09/12 – Cleandho: - Preparation of LB culture media - Inoculum of six MR6 clones 05/09/12 – Cleandho and Débora: - Digestion of pSB1K3, pSB1C3, 11J and 16P - Ligation of MR6 assembly using 3 proportions of inserts: backbone: 1:1, 3:1 and 1:3 06/09/12 – Cleandho: - Transformation of MR6 assembly in three molar proportions. 12/09/12 – Cleandho and Lucas: - Electrophoresis for reconfirmation of MR6 assembly. Result: MR6 is correct MR7
14/08/12 – Lucas - Ligation of assembly MR7 using pSB1K3 24/08/12 – Débora: - Transformation of MR7 assembly 28/08/12 – Débora and Lucas: - Plasmid extraction of MR7 clones - Digestion of MR7 clones with XbaI and PstI 29/08/12 – Débora and Lucas: - Electrophoresis of two clones of MR7 assembly. Result: failed
MR9
14/08/12 – Amanda - Transformation of MR9 assembly 27/08/12 – Lucas: - Plasmid extraction for MR9 clones - Digestion of MR9 clones with EcoRI and SpeI - Electrophoresis of MR9 clones. Result: failed 29/08/12 – Débora and Lucas: - Transformation of MR9 assembly
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.
