Team:USP-UNESP-Brazil/Notebook

From 2012.igem.org

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!align="center"|[[Team:USP-UNESP-Brazil/Attributions|Attributions]]
!align="center"|[[Team:USP-UNESP-Brazil/Attributions|Attributions]]
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Greg's lab diary (separated by assembly)
 +
 +
====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
 +
 +

Revision as of 21:14, 15 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.

Retrieved from "http://2012.igem.org/Team:USP-UNESP-Brazil/Notebook"