Team:Gunma/Experiments
Experiments
Protocols
★Incuvation
Add 5 mL LB in a 50 mL falcon tube
Add Cam(34 μg/μL) into the LB
Pick colonies by pipette and put it in the LB,repeat pipetting more than 5 times
Incubate at 37℃ in a incubator overnight
NOTE: The procedures except incuvation in a incubator are conducted in clean bench.
★Plasmid Extration(mini-prep)
Add 5 mL cultured solution into 50 mL falcon tube.
Centrifuge at 3500 rpm for 10 min at 4 ℃
Discard sup
Add 250 μL Resuspension buffer
Voltex 30s
Dispense sup into new 1.5 mL tubes
Add 300 μL lysis Buffer
Immediately add 300 μL neutralization Buffer
Invert
Centrifuge at 15000 rpm for 20 min at 4 ℃
Dispense saps into plasmid extraction column in tube
Centrifuge at 3500 rpm for 2 min at 4 ℃
Discard filtrate
Add 750 μL wash buffer
Centrifuge at 3500 rpm for 2 min at 4 ℃
Discard filtrate
Centrifuge at 15000 rpm for q0 min at 4 ℃
Put the column to a new 1.5 mL tube
Add 15 μL Elution buffer
Leave for 1 min at room temperature
Centrifuge at 15000 rpm for 10 min at 4 ℃
NOTE: We used QUAGEN P1,P2,N3,PE,EB Buffers for all plasmid extraction experiments we implemented.
★Gel Electrophoresis
Prepare X0.5 TBE buffer
Prepare X0.5 TBE 1% (2%) agarose gel
Mix sample:loading buffer:DNA fluorescent dye = 10:2:1
Apply 5 μL DNA ladder and Sample mix into the wells
Run at 100 V till the markers comes 1 cm from the edge of gel.
Check bands under UV light
Take picture if needed
NOTE: We used Midori Green instead of EtBr for all plasmid extraction experiments we implemented. Midori Green is not cancer-causing unlike EtBr, so it is safer and desireble for experiment conductors. Also we learned to prepare the agarose gel <0.5 cm tick according to the guideline of Midori Green.
★Gel Extraction
Cut bands in gel carefully and put it into 1.5 mL tube
Weigh gel to be <300 mg
Add 500 μL binding buffer
Voltex 30s
Flash
55 ℃ 15 min in Heat Block
Voltex 15s
Flash
Pipette the solution into an extraction column in a 1.5 mL tube
Centrifuge at 15000 rpm for 1 min at 4 ℃
Discard filtrate
Add 500 μL wash buffer
Centrifuge at 15000 rpm for 2 min at 4 ℃
Discard filtrate
Add 500 μL wash buffer
Centrifuge at 15000 rpm for 2 min at 4 ℃
Discard filtrate
Centrifuge at 15000 rpm for 2 min at 4 ℃
Put the column into a new 1.5 mL
Drop 30 μL Elution buffer
Hit the tube upon the desk lightly,a couple of times
Leave it for 2 min
Centrifuge at 15000 rpm for 2 min at 4 ℃
★PCR(Inverse PCR)
Prepare the PCR reaction mixture in PCR tube
Total volume: 50 μL or 100 μL
MQ water : 50-16-*
Buffer x10 : 5
dNTP : 5
MgSO4 : 3
F Primer : 1
R Primer : 1
Plasmid : 1*
KOD : 1
Total 50μL
NOTE :The volume of MQ water is dependant on the concentration of Plasmid.
Put the tubes into PCR thermal cycler
Run 29~35 cycles
98 ℃ 2.5 min
Tm ±1,2 ℃ 30s
68 ℃ 9 min
Put it on ice till Gel Electrophoresis and Gel Extraction
★In-Fusion (Ligation)
Set Heat Block at 50 ℃
Prepare the In-Fusion reaction mixture in a 1.5 mL tube
MQ water : 4 μL
Insert : 2 μL
Vector : 2 μL
(Both above are gel extracted or cloning enhanced PCR products)
In-Fusion Premix : 2 μL
Total 10 μL
Tap the tube lightly 3 times
50 ℃ 15 min in Heat Block
Put it on ice till Transformation
★Transformation
Thaw competent cell tube on ice
Add 5 μL In-Fusion product (Plasmid) into the tube
30 min on ice
42 ℃ 1 min heatshock
5 min on ice
Softly tap the tube a couple times by a finger
Put the competent cell into agarose gel plate with antibiotics
e.g. one of two of Cam 34 μg/mL, Amp 50 μg/mL, Kan μg/mL
Incubate overnight(13~16 hours) at 37 ℃ in incubator
NOTE: All the process of Transformation is conducted in cleanbench. We sprayed 70% EtOH to our hands in nitril groves, bare arms with sleeves rolled up and all devices we use inside cleanbench before we start.
★Liquid culture
This is the easiest one!😃
Add 4 g LB broth into a 200 mL Erlenmeyer flask and mess up to 200 mL with RO water
Cover the spout of flask with alminum foil and put on a piece of autoclave tape
Autoclave, 121 ℃, 20 min
★Preparation of reagents
---- 100 mM IPTG (10 mL) ----
Add 2 g Isopropyl-β-D(-)- thiogalactopyranoside(IPTG) into a falcon tube
Add MQ water 8 mL to desolve IPTG
Mess up to 10 mL
Filter sterilization
Store at -20 ℃ in 1.5 mL tubes
---- x10 TBE buffer (50mL) ----
Tris 0.89 M 5.39 g
EDTA-Na2-salt 0.02 M 0.37 g
Boric acid 0.89 M 2.75 g
Mess up to 50 mL with MQ water in 50 mL graduated cylinder.
NOTE: We poured the solution into graduated cylinder to mess up after all the particles are completely dessolved.
< Preliminary Experiments >
(0)Characterization of http://parts.igem.org/Part:BBa_I746909
See Contribution:
See BBa_I746909 part resistry experience page:
(1)Culture BL21(DE3)pLysS in hypotonic NaCl solution
Cultured 100 μL of BL21(DE3)pLysS in 200 mL LB medium with 34 μg/mL at 37 ℃ 128 rpm
When OD600 = 0.277, divided into seven 15 mL falcon tubes
Centrifuged at 3000 rpm, 5 min
Discarded sup
Added 300 mM NaCl sol. and MQ water to be 150,100,50,25,10,5,0 mM, total 6 mL
Measured OD600 every 30 min for 3 hours
See Results
Summary: BL21(DE3)pLysS significantly lessen its OD600 value when cultured under 25 mM hypotonic solution.
(2)Culture BL21(DE3)pLysS in hypotonic NaCl solution with 50 μg/mL T7 RNA polymerase
Cultured 100 μL of BL21(DE3)pLysS in 200 mL LB medium with 34 μg/mL at 37 ℃ 128 rpm
When OD600 = 0.277, divided into seven 15 mL falcon tubes
Centrifuged at 3000 rpm, 5 min
Discarded sup
Added 300 mM NaCl sol. and MQ water to be 150,100,50,25,10,5,0 mM and 50 μg/mL RNA polymerase, total 6 mL
Measured OD600 every 30 min for 3 hours
See Results
Summary: BL21(DE3)pLysS significantly lessen its OD600 value when cultured under 25 mM hypotonic solution. However adding RNA polymerase obviously inhibited it.
< Main Experiments >
We designed the DNA sequenses and primers to make our basic part and composite part.Amino acid information coding magainin-2 is gained from NICB.
DNA extracted by kitchen detergent, contact lens solution, NaCl solution
We added ATG to the sequense to enable it express and chose E.coli optimized codons for each amino acid, and added linker sequenses since we used In-Fusion to combine our insert to pSB1C3.
DNA extracted by kitchen detergent, contact lens solution, NaCl solution
5'GFP and 5'RBS were cut in inverse PCR and then RBS sequense were added as additional coding sequense of mag-2. Mag-2 is combined to the linearized pSB1C3 BBa_I746909 at the same time and was the plasmid we wanted.
http://parts.igem.org/Part:BBa_I746909
In this was our new part was generated between RBS and GFP.
http://parts.igem.org/Part:BBa_K3050000
The insert DNA and primers were synthesized by FASMAC,Japan.
http://fasmac.co.jp/
We transformed pSB1C3 BBa_K3050000 into BL21(DE3),cultured on LB agarose plate with Cam 34 μg/mL for 21 hours and picked two colonies, assigned a tube to each colony incubated in LB medium with Cam 34 μg/mL for 8 hours.
We did a fast check to know if our part is successfully expressing. We directly putted 300 μM IPTG on the colonies and we could see them emitting obvious green fluorescent light after only incuvated for an hour.
The cultured solution was diluted to be OD600 = around 0.5 in four 50 mL falcon tube in total, 8 mL and added 500 μM IPTG. After incuvated for 4.5 hours at 37 ℃, 220 rpm it obviously looked green too.
Abs600 and GFP Fluorescent light were measured several times by Perkin Elmer,Multimode Plate Reader Enspire.
See Characterization
See Results
Measurement 1-1 Before centrifuge in LB medium
Measurement 1-2 Sup after centrifuge
Measurement 2-1 Before centrifuge, in LB medium
Measurement 2-2 After ppt dissolved to MQ water
Measurement 2-3 Before centrifuge
