Difference between revisions of "Team:CCA San Diego/protocols"

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Protocols

Here we can talk about protocols in the lab

Transformation with Digest

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Digest Ligations

Materials

T4 DNA Ligation Buffer. Invitrogen, Cat No. 46-0114

T4 DNA Ligase, Thermo Scientific, Cat No. K1231

Reagent grade water, NERL, Cat No. 98555

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Methods

Set ligation as shown below in 1.5 mL tube.

The tubes were incubated at room temperature for 1 hour.

Turn on incubator-shaker at 37°C.

The tubes were then transferred to ice.

Ligation Condition (with insert)

Component Volume (μl)

Reagent grade water 4.5 μl

10X T4 ligation buffer 1.0 μl

EcoR1-Pst1
Linearized - pSB1C3 0.5 μl

EcoR1/SpeI - Linearized –promoter
(For us: BBa_J23100, BBa_J23101, BBa_J23110) 2.0 μl

XbaI/Pst1 - Linearized -Insert (Catabolic pathway)
(For us: Fluorene 1, or Fluorene 2, or Phenanthrene 1, or Phenanthrene 2) 1.5 μl

T4 DNA Ligase (5Weiss/μl) 1.5 μl

Control Ligation Condition (no insert)

Component Volume (μl)

Reagent grade water 8.0 μl

10X T4 ligation buffer 1.0 μl

EcoR1-Pst1 pSB1C3 0.5 μl

T4 DNA Ligase (5Weiss/μl) 0.5 μl

OD Calibration

Materials

1 mL LUDOX (provided in kit)

Water

96 well plate with flat with transparent bottom

Methods

Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)

Add 100 μl of H 2 O into wells A2, B2, C2, D2 (or 1 mL H 2 O into cuvette)

Measure absorbance 600 nm of all samples in all standard measurement modes in instrument

Record the data in the table below or in your notebook

Import data into Excel ( OD600 reference point tab )

Biotransformation Measurement

Materials

Cultures of clones in transformed in Escherichia coli strain BL21(DE3)

LB (Luria Bertani) media

Minimal medium

Testing substance (Fluorene and Phenanthrene)

Appropriate Antibiotics (Chloramphenicol, Tetracycline and Ampicillin)

IPTG

Baffled flasks

50 mL Falcon tube with vented cap

Incubator at 37℃

1.5mL Eppendorf tubes for sample storage

Ice bucket with ice

Pipettes

96 well plate with flat transparent bottom devices

Shaker

Instrumental Methods

Set instrument to read OD600 (as OD calibration setting)

Measure OD600 of the overnight cultures

Record data in notebook

Import data into Excel Dilution Calculation

Dilute the cultures to a target OD 600 of 0.02 (see the volume of preloading culture and media in Excel) (Dilution Calculation Sheet_1) in 4 mL LB medium + correct antibiotic combinations (for us Chloramphenicol or tetracycline or both) in 14 mL falcon tube

Incubate the cultures at 37℃ and 220 rpm

Take 450 μL samples of the cultures at various time points. At each time point, take a sample from each clone, two colonies per clone

Place samples on ice.

At the end of sampling point, measure samples

Use the same instrument setting for all time point

Pipette 100 μL of each sample into each well.

DNA Preparations

Materials

Promoters (100, 101, 110):
Reagent grade water

The promoter areas were designed by CCA-IGEM-Team 2017.

The promoter areas were synthesized by IDT.

The synthetic promoter areas were delivered to us lyophilized.

Polycistronic codons (F1, F2, P1, P2):
Reagent grade water

The mini-genes were designed by CCA-IGEM-Team 2017.

The mini-genes were synthesized by Genscript.

The genes were delivered to us lyophilized.

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

The vials containing the lyophilized DNA(~4 µg) were spun down before opening the vials for the first time

16 µL of 0.2µm filtered water was added to the lyophilized powder using a P20 pipet.

After closing the tubes, they were vortexed for 2-3 minutes and were allowed to sit at 60-65°C for 15 minutes to resuspend the DNA. The tubes were then spun.

Aliquots were taken to start cloning.

All stocks are stored at -20°C.

Gel

Materials

Agarose

1xTAE

Microwave

EtBr

Tray and comb

Methods

Measure 1 g of agarose

Mix agarose powder with 100 mL 1xTAE in a microwavable flask.

Microwave for 1-3 min until the agarose is completely dissolved

Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins.

Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL

Pour the agarose into a gel tray with the well comb in place.

Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.

Gel Purification

Materials

Finished Gel

Microtube

DNA Purification Kit

Methods

Once you have run your gel, move it to an open UV box.

Remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.

Place the gel in a labeled microfuge tube.

Using a scale, weigh the tube with the gel fragment. The weight of the gel is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.

Isolate the DNA from the gel using any gel purification kit. Follow kit instructions.

Creating Constructs

Materials

DNA samples

Transformation efficiency DNA, specific plasmid (for us: pUC 19, 10 pg/ µL), Lucigen, Cat No. F92078-1

LB Carbenicillin 50 agar plates, Cat No. Teknova, L1801

E coli BL21 DE3, Life Technology, Cat No. 60106-1

SOC (Recovery Medium), Lucigen, Cat No. F98226

15 mL culture tube

Vortex

Tooth pick

Incubator shaker

42°C Water bath

Ice and ice bucket

Pipet

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Transform 1 µL of DNA of plasmid (for us: pUC19) as transformation efficiency control

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bathat 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOCmedium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 2 volumes (50 µL and ~100µL) of the mixture onto 2 different plates of LB agar plates supplemented with Carbenicillin 100 µg/mL

Incubate plates at 37°C overnight

Count colonies and estimate transformation efficiency

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Cloning with pUC Plasmid of Lac Operation (CCA_San_Diego Specific)

Materials

Synthetic DNA
Synthetic flnB, dbfA1, dbfA2: 3219 bp

Synthetic flnE, flnD1, ORF16, flnC: 3545 bp

Synthetic phnF, phnE, phnC, phnD: 4227 bp

Synthetic phnAc, phnAd, phnB: 3174 bp

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Cloning with Constructive Promoter of different Strengths (CCA_San_Diego Specific)

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Cloning for depository (CCA_San_Diego Specific)

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Clone verification

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

Glycerol Stock

Materials

DNA miniprrep

LB agar plates, Cat No. Teknova, appropriate antibiotic

DH5a competent cells, Invitrogen, Cat 18265-017

SOC (Recovery Medium), Lucigen, Cat No. F98226

1.5 mL tube

Vortex

Pipet and tips

Ice bucket and ice

Water bath (42°C)

Incubator (37°C) and shaker

Methods

Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells

For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells

Turn on incubator-shaker at 37°C.

Turn on incubator for plates at 37°C.

Set up water bath at 42°C.

Bring to room temperature S.O.C medium.

Bring LB plates supplemented with appropriate antibiotic at room temperature.

Thaw competent cells on ice.

Aliquots competent cells in as many tubes as needed.

Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix

Incubate on ice for 20 minutes

Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.

Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube

Incubate in shaker at 37°C, 225 rpm for 30 min

Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)

Incubate plates at 37°C overnight

Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion

email igemcca@gmail.com

Canyon Crest Academy iGEM 2017 CC;