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Plasmid Plug&Play

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Associative Memory
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    Introduction

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Contents 1 Experiments 2 Plasmid Plug&Play 3 Associative Memory Network 3.1 General experiments 3.2 Assembly experiments 3.2.1 MR2 3.2.2 MR3 3.2.3 MR6 3.2.4 MR7 3.2.5 MR9

Experiments

Plasmid Plug&Play

The participation of the people related to the Plug&Play project is show in the Experiments page, it specifies which experiment was performed by each person and the moment (weeks) that it took to be performed.

https://2012.igem.org/Team:USP-UNESP-Brazil/Plasmid_Plug_n_Play/Experiments

Associative Memory Network

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

 

Assembly experiments

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