Team:BIOSINT Mexico/lab records

From 2014.igem.org

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<p>Electrophoresis gel of summer minipreps</p>
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Most of the DNA from summer minipreps was lost. Probably, due to wrong storage conditions. </p>
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Revision as of 11:55, 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


*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

Preparation of MS media for Arabidopsis thaliana

For 500 ml of MS (Murashige-Skoog) media measure:

  • 5 g of sucrose
  • 2.2 g of basal MS salt (Sigma-Aldrich)
  • 4 g of agar
  • 5 ml of Vitamin stock mix (Gamblor stock solution)
  • 500 ml of distilled water

  1. Mix all the components, except the agar, in constant agitation.
  2. Adjust the pH to 5.7 ± 0.1.
  3. Add the agar and mix gently and heat until it reaches 65 – 70 °C. (Do not boil or autoclave)
  4. Pour the agar in plates, let them cool and store at 4 °C.

Preparation of Antibiotic stocks

  • Cloramphenicol Stock:

  1. Weight 10 mg of lyophilized antibiotic for each ml of stock solution.
  2. Dissolve in ethanol and mix gently (It is possible to use Vortex)

  • Kanamicin Stock:

  1. Weight 50 mg of lyophilized antibiotic for each ml of stock solution.
  2. Dissolve in distilled water and mix gently (It is possible to use Vortex).

  • Ampicilin Stock:

  1. Weight 100 mg of liophylized antibiotic for each ml of stock solution.
  2. Dissolve in distilled water and mix gently (It is possible to use Vortex).
  3. For each 2 ml of media use 1 ul of stock antibiotic.

    Therefore the working concentration for each antibiotic (for use in and Agrobacterium tumefaciens and E. coli) will be:
  1. Cloramphenicol: 5ul/ml
  2. Kanamicin: 25 ul/ml
  3. Ampicilin: 50 ul/ml

CaCl2 Competent Cells

  1. Streak out frozen glycerol stock of bacterial cells (Top10, DH5α, etc.) onto an LB plate (no antibiotics since these cells do not have a plasmid in them). Work sterile. Grow plate overnight at 37°C.
  2. Make sure to autoclave 1 L LB (or your preferred media), 1 L of 100 mM CaCl2, 1 L of 100 mM MgCl2, 100 mL of 85 mM CaCl2, 15% glycerol v/v, 4 centrifuge bottles and caps, lots of microfuge tubes
  3. Chill overnight at 4°C 100 mM CaCl2, 100 mM MgCl2, 85 mM CaCl2, 15% glycerol v/v
  4. Prepare starter culture of cells
  5. Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or your preferred media – no antibiotics). Grow culture at 37°C in shaker overnight.
  6. For the next day, inoculate 1 L of LB media with 10 mL starter culture and grow in 37°C shaker.
  7. Measure the OD600 every hour, then every 15-20 minutes when the OD getsabove 0.2.
  8. When the OD600 reaches 0.35-0.4, immediately put the cells on ice. Chill the culture for 20-30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.
  9. (Spin #1) Split the 1 L culture into four parts by pouring about 250 mL into ice cold centrifuge bottles. Harvest the cells by centrifugation at 3000g for 15 minutes at 4°C.
  10. Decant the supernatant and gently resuspend each pellet in about 100 mL of ice cold MgCl2. Combine all suspensions into one centrifuge bottle. Make sure to prepare a blank bottle as a balance.
  11. (Spin #2) Harvest the cells by centrifugation at 2000g in the refrigerated centrifuge (~3000 rpm) for 15 minutes at 4°C.
  12. Decant the supernatant and resuspend the pellet in about 200 mL of ice cold CaCl2. Keep this suspension on ice for at least 20 minutes. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
  13. (Spin #3) Harvest the cells by centrifugation at 2000g (~3000 rpm) for 15 minutes at 4°C. At this step, rinse a 50 mL conical tube with ddH2O and chill on ice.
  14. Decant the supernatant and resuspend the pellet in ~50 mL of ice cold 85 mM CaCl2, 15% glycerol. Transfer the suspension to the 50 mL conical tube.
  15. (Spin #4) Harvest the cells by centrifugation at 1000g (~2100) for 15 minutes at 4°C.
  16. Decant the supernatant and resuspend the pellet in 2 mL of ice cold 85 mM CaCl2, 15% glycerol. The final OD600 of the suspended cells should be ~200-250.
  17. Aliquot 50 μL into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.

Heat Shock Transformation of E. coli

Note: Never vortex competent cells. Mix cells by gentle shaking.

  1. Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
  2. 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.
  3. Mix gently by flicking the tube.
  4. Chill on ice for 10 minutes. (Optional)
  5. Heat shock at 42 °C for 50 seconds.
  6. Incubate on ice for 2 minutes.
  7. Add 200 ul LB, Terrific or SOC medium and recover the cells by shaking at 37 °C.
  8. The recovery time varies with the antibiotic selection.
    • Ampicillin: 15-30 minutes
    • Kanamycin or Spectinomycin: 30-60 minutes
    • Chloramphenicol: 60-120 minutes
  9. 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.
  10. Incubate at 37 °C. Transformants should appear within 8 – 16 hrs.

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