Team:BIOSINT Mexico/lab records
From 2014.igem.org
(Difference between revisions)
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<h4>Electrophoresis gel of Miniprep</h4> | <h4>Electrophoresis gel of Miniprep</h4> | ||
+ | |||
+ | <h3>8 september </h3> | ||
+ | <h4>E.coli top 10 inoculation </h4> | ||
+ | <b>Procedure:</b> | ||
+ | <h4>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 </h4> | ||
+ | |||
+ | <h4>Plasmid rehydratation of Agrobacterium vectors from paper filter. </h4> | ||
+ | <h3>9 september </h3> | ||
+ | <h4>E.coli top10 Electrocompent cells </h4> | ||
+ | <b>Procedure:</b> | ||
+ | |||
+ | <br>*Dilute the cells 100X. 100 ml LB Amp. Grow at OD600 between 0.6 and 0.8. | ||
+ | <br>*Split the culture into 2x 50 ml falcon tubes, on ice. | ||
+ | <br>*Centrifuge at 4 °C for 10 min at 4000 rpm. | ||
+ | <br>*Wash and combine the cells. Remove the supernatant. | ||
+ | <br>*Resuspend the cells in 2x 25 ml of ice cold water. Combine the volumes | ||
+ | <br>*Wash the cells 2 more times and entrifuge at 4 °C for 10 min at 4000 rpm. | ||
+ | <br>*Resuspend in 50 ml of ice cold water and Repeat. | ||
+ | <br>*Wash and concentrate the cells and Centrifuge at 4 °C for 10 min at 4000 rpm. | ||
+ | <br>*Resuspend in 1-2 ml of ice cold water and storage at -20°C | ||
+ | <h3>10 september </h3> | ||
+ | <h4>Electro-Transformation of RBS, VP16, Pif6, Pif6 (2) and TetR</h4> | ||
+ | <b>Procedure:</b> | ||
+ | |||
+ | <br>*Add 6 μl DNA to tubes containing 50 μl electrocompetent cells. Homogenize. | ||
+ | <br>*Transfer mixture to a prechilled cuvette. Insert the cuvette into the device. | ||
+ | <br>*******Electroporation: | ||
+ | <br>*******Mode Prokaryotes | ||
+ | <br>*******Voltage (V) 1,700 V | ||
+ | <br>*******Time constant (τ) 5 ms | ||
+ | <br>*Add 1 ml SOC medium and transfer to a tube. Incubate 60 minutes with shaking at 37 °C. | ||
+ | <br>*Plate on LB plates with Ampicillin. | ||
+ | <h3>11 september </h3> | ||
+ | <h4>Inoculation of E.coli with RBS, VP16, Pif6, Pif6 (2) and TetR in Terrific broth</h4> | ||
+ | <b>Procedure:</b> | ||
+ | <br>*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 | ||
+ | |||
+ | <h3>12 september </h3> | ||
+ | <h4>Miniprep: RBS, VP16, Pif6, Pif6 (2) and TetR</h4> | ||
+ | <b>Procedure:</b> | ||
+ | <br>*Centrifugue E,coli tramsformed culture at 12000 rpm for 1 minute two times | ||
+ | <br>*Resuspend in 250 µl resuspension buffer | ||
+ | <br>*Add 250 µl of Lysis Buffer L7 (room temperature) | ||
+ | <br>*Add 350 µl of precipitation Buffer N4 | ||
+ | <br>*Centrifuged at 12000 rpm for 10 minutes and tansfer the supernatant to a spin column | ||
+ | <br>*Centrifuge at 12000 rpm for a minute | ||
+ | <br>*The supernatant was discarded and 500 µl of Wash Buffer were added | ||
+ | <br>*The column was centrifuged at 12000 rpm for 1 minute | ||
+ | <br>*700 µl of Wash Buffer W9 with ethanol were added to the column | ||
+ | <br>*The column in the washtube was centrifuged at 12000 rpm for 1 minute | ||
+ | <br>*75 µl of preheated TE Buffer were added (65°C preheated) | ||
+ | <br>*The column was centrifuged at 12000 rpm for 2 minutes | ||
+ | |||
+ | <h4>Electrophoresis Gel of miniprep [RBS, VP16, Pif6, Pif6 (2) and TetR]</h4> | ||
+ | |||
+ | <h3>13 september </h3> | ||
+ | <h4>Restriction digest of MerB, MerE and Psc1c3</h4> | ||
+ | <b>Procedure:</b> | ||
+ | <br>*Add 9 ul of DNA to be digested, and 6 ul of dH20 | ||
+ | <br>*Pipet 2.5ul of NEB Buffer 2 to each tube. | ||
+ | <br>*Pipet 0.5ul of BSA to each tube. | ||
+ | <br>*In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of PstI. | ||
+ | <br>*In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, | ||
+ | <br>*Incubate the restriction digests at 37°C for 60 minutes | ||
+ | |||
+ | <h4>Electrophoresis gel: Restriction digest of MerB, MerE</h4> | ||
+ | |||
+ | <h3>14 september </h3> | ||
+ | <h4>Restriction digest of MerB, MerE and Psc1c3 [NEW ENZYMES]</h4> | ||
+ | <b>Procedure:</b> | ||
+ | <br>*Incubate the restriction digest at 37°C for 60min, and then 67°C for 20min | ||
+ | |||
+ | <b><br>Notes:</b> | ||
+ | |||
+ | <h4>Electrophoresis gel: Restriction digest of MerB, MerE</h4> | ||
Revision as of 19:15, 17 October 2014
Lab Records
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
Electrophoresis gel: Ligation of Test 2
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
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
Heat Shock Transformation of E. coli
Note: Never vortex competent cells. Mix cells by gentle shaking.
- Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
- Place 20 ul of cells in a pre-chilled Eppendorf tube.
- For an Intact Vector: Add 0.5 ul or less to the chilled cells
- For a Ligation Product: Add 2-3 ul to the chilled cells.
- Mix gently by flicking the tube.
- Chill on ice for 10 minutes. (Optional)
- Heat shock at 42 °C for 50 seconds.
- Incubate on ice for 2 minutes.
- Add 200 ul LB, Terrific or SOC medium and recover the cells by shaking at 37 °C.
- The recovery time varies with the antibiotic selection.
- Ampicillin: 15-30 minutes
- Kanamycin or Spectinomycin: 30-60 minutes
- Chloramphenicol: 60-120 minutes
- Plate out the cells on selective LB. Use glass beads to spread the cells. The volume of cells plated depends on what is being transformed.
- For an Intact Vector: High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.
- For a Ligation Product: Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.
- Incubate at 37 °C. Transformants should appear within 8 – 16 hrs.