Team:NTU-Taida/Notebook/Protocol
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Protocol
Here are all the protocols we have used in our experiments.
Transformation
- Add dd water 20 ul into biobrick kit, pipette and extract.
- Prepare competent cells on ice.
- Mix 100 ul of competent cells with 15 ul plasmids (1 ul for biobrick kit extract) in eppendorfs and put on ice for 30 mins.
- Heat shock 42oC 90 secs and put them on ice for 3 mins.
- Add LB broth 1000 ul to the eppendorfs.
- Put on ice for 2 mins.
- Incubate at 37oC for 20 mins.
- Centrifuge for 2 mins. Drop the supernatant, and resuspend the cells with remaining medium.
- Spread the cells on LB agar plates with appropriate antibiotics and incubate at 37 oC for 18 hrs.
E coli culture
- Add 3mL LB/Amp (50 μl/mL) in each tube
- Pick up plate and use tip to scrape a single colony
- Soak the colony inside the incubation tube
- Put the tubes on the shaker of 37℃ and incubate overnight(16hr)
Conventional assembly
Digestion
- Calculate the amount of each reagent { total 15 ul (vector 20ul): two RE 0.7ul/10Xbuffer 1.5ul or 1.5 ug
(vector 2ul)/DNA=1000ng/ddH2O=remaining}
- Adding each reagent ddH2O10XbufferDNARE
(37℃ incubator) After 2 hr digestion, put all the samples to go electrophoresis.
Electrophoresis preparation
- Agarose gel synthesis:
1% Agarose: 0.8g agarose +80 ml TAE buffer
- Mix and heat in microwave oven until boiling for about 2 minutes. (If EtBr is necessary, it is added after this step)
- The mixture is added into the gel plate to create one 40ml well and two 20ml well.
- 20 minutes is required to form the gel.
Electrophoresis
- DNA mixture is loaded add loading dye (1.5 ) is added into the eppendorf.
- Load the dye mixture into the well, as well as the marker (general ruler, preserved in 4∘C fridge, upper right) and start to electrophoresis for 30 minutes.
Results
- Add SYBR dye (must be protected from light, packed in an aluminum foil)
- Add 5 of 10000X SYBR Safe to TAE solution and swirl the container gently to mix. (SYBR safe stock is on 4∘C refrigerator)
- Sock the gel in the container; ensure the gel is fully immersed during staining.
- Incubate for 15~20 minutes (continuously agitate the gel on an orbital shaker at 50rpm)
- ※If the signal is not strong enough, the gel can be stained longer or the staining solution has to be replaced.
Gel extraction
- Cut the insert and vector from the gel with blade
- Put into gel extraction column and centrifuge 13,000 rpm for 30 sec
- Use the elution samples for DNA ligation
Ligation
- vector 0.5 μl
- insert 1 μl
- ligase buffer 1.5 μl
- ligase 0.5 μl
- ddH2O 11.5 μl
- Place under room temperature for about 3 hr
(Place under 16℃ and react for 16 hours) (Stock at -20℃ refrigerator, if it would not be dealt right away)
Transformation
- Competent cells (100ul) were mixed with plasmids (15ul)(1ul for biobrick kit extract) in eppendorfs and put on ice for 30 mins.
- They were under 42oC heat shock for 90 seconds and put on ice for 3 mins.
- Luria-Bertani (LB) broth (1000ul) was added to the eppendorfs.
- Cells were incubated at 37oC for 20 mins.
- They were centrifuged for 2 mins. The supernatant was dropped, and the remaining medium was pipetted to resuspend the cells.
- Cells were spread on LB agar plates with ampicillin, incubated for 18 hours. (37oC)
E.coli culture
- Add 3mL LB/Amp (50 μl/mL) in each tube
- Pick up plates and use tip to scrape a single colony
- Soak the colony inside the incubation tube (each plasmids 5 tube)
- Put the tubes on the shaker of 37℃ incubate, overnight(16hr)
Selection
- Spin down 1.0ml of E. coli overnight cells in a microcentrifuge. (13,000 rpm, room temp., 30~45 sec) and remove medium.
- Add 50μl of TEN (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 100mM NaCl)
- Vortex for 2 min. Turn upside down for 5 times. (VWR multivirtexer)
- Add 70μl phenol/chloroform/isoamyl alcohol (25/24/1, with 1% β-ME)
- Vortex for 2~3 seconds, microcentrifuge for 10 min.
- Transfer upper solution 70 μl (without touching the interface) to new microcentrifuge tube.
- Add 20μl of NH4OAc (7.5M) and 100μl of isopropanol (room temp.)
- Vortex for 5 sec, microcentrifuge for 1 min (room temp.)
- Wash the pellet with 70% EtOH(1ml) and dry it completely.
- Use a paper towel to help dry the alcohol.
- Re-suspend the pellet in 15μl of ddH2O and 1/50 RNase.
- Add the ddH2O an d RNase together before adding into the eppendorf.
Digestion (check)
- samples for each of 7 kinds DNA
- Enzyme: EcoR I, Pst I, add 0.2 microliter to each reaction(15 microliter)
- DNA: 5 microliter (3 if already done minipreparation)
- Add 10X buffer, ddH2O, enzymes, DNA together
- Digest in 37 degree incubator for 1.5 hrs
3A Assembly
Restriction Digest
Materials
- Your two part samples: Miniprepped DNA (in BioBrick RFC[10] plasmid backbones)
- Linearized plasmid backbone (with a different resistance to the plasmid backbones containing your part samples)
- EcoRI, XbaI, SpeI, PstI
- NEB Buffer 2
- BSA
- dH20
Procedure
Digest Reaction
- dH20
- NEB Buffer 2
- BSA
- DNA and Enzymes
- Digest Part A with EcoRI and SpeI
- Digest Part B with XbaI and PstI
- Digest linearized plasmid backbone with EcoRI and PstI
- Combine 1 ul of each restriction digest reaction with 1 ul of ligase in a 25 ul reaction.
- Transform the ligation product.
- If the input parts are good, almost all colonies will be correct.
- If desired analyze the transformation with single colony PCR followed by agarose gel electrophoresis.
- Miniprep clones that generated a band of the appropriate size.
- Sequence the clone.
- Record the sequence information in the Registry.
DNA Minpreparation
Tips for 10 min plasmid DNA minipreparation
- Grow 2.0 ml overnight culture
- Spin down 1.0 ml if E. coli overnight cells in a microcentrifuge (13,000 rppm, room temp., 30-45 sec) and remove medium.
- Add 50 ul of TEN (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 100mM NaCl)
- Vortex for 2 min. (VWR multivortexer)
- Add 70 ul phenol/chloroform/isoamyl alcohol (25/24/1, with 1% β-ME)
- Vortex for 2-3 sec, microcentrifuge for 10 min (room temp.)
- Transfer upper solution 70 ul (without touching the interface) to new microcentrifuge tube
- Add 20 ul of NH4OAc (7.5M) and 100 ul of isopropanol (room temp.)
- Vortex for 5 sec, microcentrifuge for 1 min (room temp.)
- Wash the pellet with 70 % EtOH (1 ml) and dry it completely.
- Resuspend the pellet in 15 ul of dd H2O or TE. and 1 / 50 RNase. Store at -20oC
Note. Use 4 ul of DNA for digestion (30-60 min in 15 ul with RNase or “express” digestion 3 X 10 sec in a microwave oven), use 18 ul for sequencing.