3 June

Team meeting

Notes: Summer lab Schedule!

4 June

Media preparation LB broth and LB agar and E.coli inoculation

Procedure:
* 14g of LB agar were weighted to prepare 400ml of medium
* 1 g of LB broth were weighted to prepare 500 ml of medium
* Autoclave at 121°C for 15 minutes
* E.coli was inoculated in petri dishes and incubated at 37°C
Notes: The growth mediums in storage were revised; they didn’t show signs of contamination

Calcium chloride for competent cells

Procedure:
* Sterilize distilled water in the autoclave for 15 minutes at 121⁰c
* Dilute 1.1159 g of calcium chloride in 50 ml of sterile distilled water
* Fill a syringe to its maximum capacity and connect a ministart 0.2µm filter
* Pour the content of the syringe into a sterile falcon tube through the filter

5 June

E.coli Competent cells

Procedure:
* Add 1.5 ml of E.coli in LB broth to Ice tubes
* Centrifuged for 3 minutes at 6000 rpm, two times
* The supernatant was discarded
* The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
* Incubated in ice for 20 minutes
* The tubes were centrifuged again for 3 minutes at 6000 rpm
* The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
* They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c

MS media preparation for Arabidopsis

Procedure:
* For 500 ml of MS media measure:5 g of sucrose, 2.2 g of basal MS salt, 4 g of agar, 5 ml of Vitamin Gamblor and 500 ml of distilled water
* Mix all the components, except the agar, in constant agitation.
* Adjust the pH to 5.7 ± 0.1.
* Add the agar and mix gently and heat until it reaches 65 – 70 °C. (Do not boil or autoclave)
* Pour the agar in plates, let them cool and store at 4 °C.

6 June

Transformation of E.coli with MerB and MerE (from DNA synthesis)

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*4 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and ampicillin(100mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:The concentration of lyophilized DNA from synthesis was 200ng, rehydrated with 1 ml of elution buffer, for a final concentration of 2ng/10 ul. E.coli inoculation from SOC media in LB dishes with ampicillin, will be done tomorrow The first colonies appear three days later. The transformation efficiency of competent cells should be review.

9 June

Growing arabidopsis seeds in media

Procedure:
Notes:

Biobricks rehydration and transformation of E.coli: PhyB, Pif6, NLS, VP16 and RFP 10 pg( transformation efficiency)

Procedure:
* Kit plates were rehydrated with 10 ul of distillated water
* 50 µl of competent cells were placed in a cold eppendorf tube
* 2 µl of DNA sample were added
* It was mixed gently and left incubating in ice for 30 minutes
* The tube was put through heat shock in water at 42⁰c for 45 seconds
* The tube was placed in ice for 2-5 minutes
* 900 µl of S.O.C. were added
* The tube was left incubating for 1 hour at 37⁰c and 120 rpm
* A dish with LB agar and Chloramphenicol (35mg/ml) was striated with the transformed cells
* The dish was sealed and left incubating at 37⁰c

Notes:The first colonies appear two days later and transformation efficiency kit is not working as expected. The transformation efficiency of competent cells should be review at different concentration.
Antibiotic concentration should be review.

10 June

Miniprep MerB and MerE (sample from DNA synthesis)

Procedure:
* Centrifuge E,coli transformed culture at 12000 rpm for 1 minute two times
* Resuspend in 250 µl resuspension buffer
* Add 250 µl of Lysis Buffer L7
* Add 350 µl of precipitation Buffer N4
* Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
* Centrifuge at 12000 rpm for a minute
* The supernatant was discarded and 500 µl of Wash Buffer were added
* The column was centrifuged at 12000 rpm for 1 minute
* 700 µl of Wash Buffer W9 with ethanol were added to the column
* The column in the washtube was centrifuged at 12000 rpm for 1 minute
* 75 µl of preheated TE Buffer were added
* The column was centrifuged at 12000 rpm for 2 minutes
Notes: Inventory season!, running gels must wait until next week

11 june

Miniprep PhyB, Pif6, NLS, VP16 and RFP 10 pg (Transformation efficiency)

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added
*The column was centrifuged at 12000 rpm for 2 minutes
Notes:Inventory season!, running gels must wait until next week

13 june

Media preparation (LB broth and LB agar)

Procedure
*14g of LB agar were weighted to prepare 400ml of medium
*1 g of LB broth were weighted to prepare 500 ml of medium
*Autoclave at 121°C for 15 minutes

17 june

Restriction digest of MerE, MerB and PSB1C3

Procedure:
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Add 2.5ul of NEBuffer 2.
*Add 0.5ul of BSA.
*Add 0.5ul of EcoRI.
*Add 0.5ul of PstI.
*Mix well and spin down briefly.
*Incubate the restriction digest at 37C for 30min, and then 80C for 20min
Notes:Mislabelling of sample, the experiment must be repeated

Ligation of MerE and MerB in PSB1C3

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample, 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C. Inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
Mislabelling of sample, the experiment must be repeated

18 june

Restriction digest of MerE, MerB and PSB1C3

Procedure:
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Add 2.5ul of NEBuffer 2.
*Add 0.5ul of BSA.
*Add 0.5ul of EcoRI.
*Add 0.5ul of PstI.
*Mix well and spin down briefly.
*Incubate the restriction digest at 37C for 30min, and then 80C for 20min

Ligation of MerE and MerB in PSB1C3

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample, 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes:T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants

Gel electrophoresis: PhyB, MerE, VP16, NLS, PIf6, MerB, RFP and PSB1C3

Procedure:
*2% agarose gel, the current was set to 100 V for 55 minutes. Results:
Notes:No band appears, there is not DNA sample in the gel. The transformation experiments must be repeated.

19 june

Gel Electrophoresis: MerB and MerE Restriction digest and ligation

Procedure:
*2% agarose gel, the current was set to 100 V for 55 minutes. Results:
Notes:No band appears, there is not DNA sample in the gel. The transformation, restriction digests and ligation experiments must be repeated.

20 june

Transformation of E.coli with MerB and MerE in PSB1C3

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Ampicillin (100mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:To confirm that the gel has done correctly, parts ligated with PSB1C3 were transformed in E,coli After one week, no colonies growth in petri dishes

23 june

Competent cells

Procedure
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
Note: The first competent cells didn’t work as expected. The same protocol was applied this time.

Test 1: Transformation of E.coli with MerB, MerE, NLS, PhyB, VP16, AGS linker, PIF6, PolyA, TetR, RFP 10 pg (transformation efficiency)

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*2 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Chloramphenicol (35mg/ml) or Ampicillin(100mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes: The next day no colony appears. A second culture of the same SOC solution was done. The first colonies of MerB, MerE from the first culture appears two days later and the next day the second culture grew. But transformation efficiency kit and parts from the distribution kit are not working as expected.
Some colonies of Pif6 and RFP grew, after three days of incubation.
Antibiotic concentration of Chloramphenicol and competent cells efficiency should be review.

Terrific Broth preparation

Procedure:
*Weight 42.5 grams of Terrific Broth (Sigma-Aldrich) for each liter of media.
*Dilute in Erlenmeyer flasks with the adequate amount of distilled water.
*Autoclave

Pasar arabidopsis a tierra y medio

24 june

Inoculation of Transformed colonies of Test 1 in terrific broth (MerB, MerE, Pif6 and RFP)

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

Media preparation: LB agar with antibiotics at different concentrations

25 june

Competent cells

Procedure:
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c
Notes: After the problems with test1, efficiency of competent cells it was not as expected. New competent cells was made.

Miniprep (1) MerB, MerE, RFP(te),Pif6

Aim of the experiment:
*Plasmid extraction from colonies of “transformation (test 1)” Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added
*The column was centrifuged at 12000 rpm for 2 minutes
Notes:

Test 2: Transformation of E.coli with MerB, MerE, NLS, PhyB, VP16, AGS linker, PIF6, PolyA, TetR, RFP(control)

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*2 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Chloramphenicol (15mg/ml) / Ampicillin (100 mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:The colonies of MerB, MerE, NLS, AGS, Pif6, VP16 and TetR from the first culture appear the next day. But transformation efficiency kit and parts from the distribution kit are not working as expected. Antibiotic concentration of Chloramphenicol was changed to (15 mg/ml).

26 june

Miniprep (2) MerB, MerE, NLS, AGS, Pif6, VP16, TetR

Aim of the experiment:

Plasmid extraction from colonies of “transformation (test 2)”

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added
*The column was centrifuged at 12000 rpm for 2 minutes

27 june

Electrophoresis Gel of MerE, MerB, RFP

Results:

30 june

Electrophoresis gel of Miniprep: NLS, AGS, Pif6, VP16, TetR

1 july

Test1: Restriction digest TetR, Pif6, MerB, NLS, PolyA, MerE, MerB, PSB1C3

Procedure
***Note: some parts are A/B, cut them with EcoRI and PstI
*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2 to each tube.
*Pipet 0.5ul of BSA to each tube.
*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
*In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.

2 july

Test 1: Ligation

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants
Electrophoresis Gel (same samples as 30 july) and more samples

3 july

Electrophoresis gel: Restriction digests gel of Test 1

Electrophoresis gel: Ligation of test 1

4 july

Test 2: Restriction digest

***Note: for parts are A/B, cut with EcoRI and PstI
*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2 to each tube.
*Pipet 0.5ul of BSA to each tube.
*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
*In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.

7 july

Test 2: Ligation

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants

8 july

Electrophoresis gel: Restriction digest of test 2

Electrophoresis gel: Ligation of Test 2

9 july

Transformation of E.coli with Test 2

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:

Rehydratation and Transformation PhyB

*Kit plates were rehydrated with 10 ul of distillated water
*50 µl of competent cells were placed in a cold eppendorf tube
*2 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

10 july

Test for antibiotic concentration

14 july

Restriction digests of linear plasmid PSB1A3

Procedure
*Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tube
*Add 10 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2
*Pipet 0.5ul of BSA
*Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
*Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture
*Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes.

Electrophoresis gel of PhyB

15 july

Competent cells

Procedure:
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c

Ligation of Test

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 4 ul of Linear vector, 8 ul of DNA sample (4ul for sample A, 4 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of distiller water.
*Incubate 1 hour at 22°C.
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use up to 5 μL of the mixture for transformation of 50 μL of chemically competent cells
Notes: T4 ligase from NEB is scarce; we replace it with thermo scientific ligase.
Reaction time was extended from ten minutes to one hour, to increase the overall number of transformants

16 july

Transformation of E.coli with

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c
Notes:

17 july

Last day of summer work!

Notes:Colonies for ligation never appear, all the minipreps were stored at -4°C to continue working on August!

18 august

Media preparation LB broth and LB agar and E.coli k.12, E.coli Top10 and agrobacterium inoculation

Procedure:
*14g of LB agar were weighted to prepare 400ml of medium
*Weight 42.5 grams of Terrific Broth for each liter of media.
*Autoclave
*E.coli was inoculated in petri dishes and incubated at 37°C
Notes:The growth mediums in storage were review; they didn’t show signs of contamination

19 august

Electrophoresis gel of summer minipreps

Notes:Most of the DNA from summer minipreps was loses. Probably, due to wrong storage conditions.

20 august

Transformation of E.coli with NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

21 august

Media preparation LB broth and E.coli Top10 inoculation

Procedure:
*14g of LB agar were weighted to prepare 400ml of medium
*1 g of LB broth were weighted to prepare 500 ml of medium
*Autoclave at 121°C for 15 minutes
*E.coli was inoculated in petri dishes and incubated at 37°C
Notes:The growth mediums in storage were review; they didn’t show signs of contamination

22 august

Calcium chloride competent cells

Procedure:
*Add 1.5 ml of E.coli in LB broth to Ice tubes
*Centrifuged for 3 minutes at 6000 rpm, two times
*The supernatant was discarded
*The pellet was gently resuspended with 1.2 ml CaCl2 0.1M
*Incubated in ice for 20 minutes
*The tubes were centrifuged again for 3 minutes at 6000 rpm
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c

Transformation of E.coli with NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

25 august

Restricking plates with summer transformations in LB agar with antibiotics: NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerE and MerB

26 august

Inoculation of Transformed colonies of summer transformations in terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

27 august

Miniprep: NLS, PhyB, VP16, PiF6, TetR, MerE, Pif6 and MerB

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of minipreps: NLS, PhyB, VP16, PiF6, TetR, MerE, Pif6 and MerB

28 august

Transformation NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerB and MerE

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

29 august

Inoculation of Transformed colonies of NLS, PolyA, PhyB, VP16, PiF6, TetR, AGS, MerE and MerB in terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

1 september

Miniprep: MerB and MerE

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel miniprep: MerE y MerB

2 september

PCR

Electrophoresis Gel: PCR products

3 september

PCR

Electrophoresis Gel: PCR products

Transformation

Procedure
*50 µl of competent cells were placed in a cold eppendorf tube
*3 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 45 seconds
*The tube was placed in ice for 2-5 minutes
*900 µl of S.O.C. were added
*The tube was left incubating for 1 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

4 september

Inoculation of Transformed colonies of PhyB, Pif6, AGS, NLS, VP16 and PhyB(2) in terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

5 september

Miniprep: PhyB, Pif6, AGS, NLS, VP16 and PhyB(2)

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep

8 september

E.coli top 10 inoculation

Procedure:

Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

Plasmid rehydratation of Agrobacterium vectors from paper filter.

9 september

E.coli top10 Electrocompent cells

Procedure:
*Dilute the cells 100X. 100 ml LB Amp. Grow at OD600 between 0.6 and 0.8.
*Split the culture into 2x 50 ml falcon tubes, on ice.
*Centrifuge at 4 °C for 10 min at 4000 rpm.
*Wash and combine the cells. Remove the supernatant.
*Resuspend the cells in 2x 25 ml of ice cold water. Combine the volumes
*Wash the cells 2 more times and entrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 50 ml of ice cold water and Repeat.
*Wash and concentrate the cells and Centrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 1-2 ml of ice cold water and storage at -20°C

10 september

Electro-Transformation of RBS, VP16, Pif6, Pif6 (2) and TetR

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
*******Electroporation:
*******Mode Prokaryotes
*******Voltage (V) 1,700 V
*******Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates with Ampicillin.

11 september

Inoculation of E.coli with RBS, VP16, Pif6, Pif6 (2) and TetR in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

12 september

Miniprep: RBS, VP16, Pif6, Pif6 (2) and TetR

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis Gel of miniprep [RBS, VP16, Pif6, Pif6 (2) and TetR]

13 september

Restriction digest of MerB, MerE and Psc1c3

Procedure:
*Add 9 ul of DNA to be digested, and 6 ul of dH20
*Pipet 2.5ul of NEB Buffer 2 to each tube.
*Pipet 0.5ul of BSA to each tube.
*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of PstI.
*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI,
*Incubate the restriction digests at 37°C for 60 minutes

Electrophoresis gel: Restriction digest of MerB, MerE

14 september

Restriction digest of MerB, MerE and Psc1c3 [NEW ENZYMES]

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min
Notes:

Electrophoresis gel: Restriction digest of MerB, MerE

DNA Gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C

15 september

Nanodrop

Ligation of MerB and MerE in Psb1c3

E. coli Electrocompetent cells

Procedure:
*Dilute the cells 100X. 100 ml LB Amp. Grow at OD600 between 0.6 and 0.8.
*Split the culture into 2x 50 ml falcon tubes, on ice.
*Centrifuge at 4 °C for 10 min at 4000 rpm.
*Wash and combine the cells. Remove the supernatant.
*Resuspend the cells in 2x 25 ml of ice cold water. Combine the volumes
*Wash the cells 2 more times and entrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 50 ml of ice cold water and Repeat.
*Wash and concentrate the cells and Centrifuge at 4 °C for 10 min at 4000 rpm.
*Resuspend in 1-2 ml of ice cold water and storage at -20°C

16 september

Electrotransformation of MerB and MerE in Psb1c3

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
*******Electroporation:
*******Mode Prokaryotes
*******Voltage (V) 1,700 V
*******Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates with Ampicillin.

17 september

Electrotransformation of MerB and MerE in Psb1c3

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
*******Electroporation:
*******Mode Prokaryotes
*******Voltage (V) 1,700 V
*******Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates with Chloramphenicol.

18 september

Inoculation in Terrific broth [MerB and MerE in Psb1c3]

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

19 september

Miniprep: MerB and MerE in Psb1c3

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel [Miniprep: MerB and MerE in Psb1c3]

20 september

Creating tools:

Media preparation LB broth and LB agar

Procedure:
*14g of LB agar were weighted to prepare 400ml of medium
*1 g of LB broth were weighted to prepare 500 ml of medium
*Autoclave at 121°C for 15 minutes
*E.coli was inoculated in petri dishes and incubated at 37°C

Stocks: Ampicillin, Kanamycin and Chloramphenicol

Procedure:
*******Chloramphenicol Stock:
*Weight 10 mg of lyophilized antibiotic for each ml of stock solution.
*Dissolve in ethanol and mix gently (It is possible to use Vortex)
*******Kanamicin Stock:
*Weight 50 mg of lyophilized antibiotic for each ml of stock solution.
*Dissolve in distilled water and mix gently (It is possible to use Vortex).
*******Ampicilin Stock:
*Weight 100 mg of liophylized antibiotic for each ml of stock solution.
*Dissolve in distilled water and mix gently (It is possible to use Vortex).
*******Working concentration for each antibiotic will be:
*Cloramphenicol: 5l/ml
*Kanamicin: 25 l/ml
*Ampicilin: 50 l/ml

Inoculation E.coli in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

21 september

E. coli Calcium Chloride competent cell protocol

*Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning and shake @ 37°C for 3hrs.
*Put the cells on ice for 10 min and collect the cells by centrifugation for 3 mins @6krpm
*Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl
*Incubate on ice x 20 mins and centrifuge 3 mins @6krpm
*Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol
*Dispense in microtubes (300μL/tube). Freeze in -80°C.

Transformation: Measure competent cells Transformation efficiency with PGLO

22 september

Restriction digest RBS(A), PhyB(B), AGS(A), VP16(B), MerE(C), MerB(C), NLS (A) and PolyA(B)

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis gel of restriction digest: RBS, PhyB, AGS, VP16, MerE, MerB, NLS and PolyA

DNA Gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C

23 september

Ligation RBS with PhyB, AGS with VP16, RBS with MerE, RBS with MerB and NLS with PolyA

Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 12 ul of DNA sample (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
*Incubate 1 hour at 22°C. (termocycle)
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells

Electrotransformation of E.coli with [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]

Procedure:
*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize.
*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device.
********Electroporation:
********Mode Prokaryotes
********Voltage (V) 1,700 V
********Time constant (τ) 5 ms
*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C.
*Plate on LB plates.

24 september

Inoculation of E.coli with [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA] in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

25 september

Miniprep [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep: [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and NLS-PolyA]

26 september

Restriction digest [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and V3]

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis Gel of restriction digest: [RBS-PhyB, AGS-VP16, RBS-MerE, RBS-MerB and V3]

DNA Gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C Ligation of RBS-MerE with RBS-MerB, RBS-PhyB with AGS-VP16 and of RBS-MerE with RBS-MerB in V3. Procedure
*Mix well and spin down briefly.
*Prepare a mixture with 12 ul of DNA sample (6ul for sample A, 6 ul for sample B), 2 ul of T4 Ligase Buffer, 1 ul of T4 ligase and 5 ul of nuclease free water.
*Incubate 1 hour at 22°C. (termocycle)
*inactivation of T4 DNA ligase at 65°C for 10 min
*Use 10 μL of the mixture for transformation of 50 μL of chemically competent cells

Transformation of E.coli and Agrobacterium with [RBS-MerE-RBS-MerB and RBS-PhyB-AGS-VP16]

Procedure:
*50 µl of competent cells were placed in a cold eppendorf tube
*10 µl of DNA sample were added
*It was mixed gently and left incubating in ice for 30 minutes
*The tube was put through heat shock in water at 42⁰c for 60 seconds
*The tube was placed in ice for 5 minutes
*200 µl of S.O.C. were added
*The tube was left incubating for 2 hour at 37⁰c and 120 rpm
*A dish with LB agar and Kanamycin (35mg/ml) was striated with the transformed cells
*The dish was sealed and left incubating at 37⁰c

27 september

Inoculation of E.coli and Agrobacterium with [RBS-MerE-RBS-MerB] in Terrific broth

Procedure:
*Inoculate 10 ml terrific broth medium with a single colony from a petri dish culture of E. coli K12. Grow overnight at 37 °C with shaking

28 september

Miniprep [RBS-MerE-RBS-MerB]

Procedure:
*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times
*Resuspend in 250 µl resuspension buffer
*Add 250 µl of Lysis Buffer L7 (room temperature)
*Add 350 µl of precipitation Buffer N4
*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column
*Centrifuge at 12000 rpm for a minute
*The supernatant was discarded and 500 µl of Wash Buffer were added
*The column was centrifuged at 12000 rpm for 1 minute
*700 µl of Wash Buffer W9 with ethanol were added to the column
*The column in the washtube was centrifuged at 12000 rpm for 1 minute
*75 µl of preheated TE Buffer were added (65°C preheated)
*The column was centrifuged at 12000 rpm for 2 minutes

Electrophoresis gel of Miniprep: [RBS-MerE-RBS-MerB]

29 september

Restriction digest of RBS-PhyB and AGS-VP16

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis Gel of Restriction digest [RBS-PhyB and AGS-VP16]

30 september

Restriction digest of RBS-PhyB and AGS-VP16

Procedure:
*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min

Electrophoresis Gel of Restriction digest [RBS-PhyB and AGS-VP16]

DNA gel extraction

Procedure
*Place 400 mg of the excised gel into a 1.5-mL tube.
*Add 3 volumes Gel Solubilization Buffer (L3) for every 1 volume of gel
*Place the tube into a 50°C water bath and at least 10 minutes.
*Add 1 gel volume isopropanol to the dissolved gel slice. Mix well.
*Pipet the dissolved gel onto a Column and Centrifuge at >12,000 × g for 1 minute.
*Add 500 μL Wash Buffer (W1) to the Column and centrifuge at >12,000 × g for 1 minute.
*Centrifuge the column at maximum speed for 2 minutes and place into a Recovery Tube.
*Add 50 μL Elution Buffer (E5), incubate for 1 minute, centrifuge at >12,000 × g for 1 minute.
*Store the purified DNA at 4°C