Team:NTU-Taida/Notebook/Protocol

Protocol

Here are all the protocols we have used in our experiments.

Transformation

1. Add dd water 20 ul into biobrick kit, pipette and extract.
2. Prepare competent cells on ice.
3. 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.
4. Heat shock 42^oC 90 secs and put them on ice for 3 mins.
5. Add LB broth 1000 ul to the eppendorfs.
6. Put on ice for 2 mins.
7. Incubate at 37^oC for 20 mins.
8. Centrifuge for 2 mins. Drop the supernatant, and resuspend the cells with remaining medium.
9. Spread the cells on LB agar plates with appropriate antibiotics and incubate at 37 ^oC for 18 hrs.

E coli culture

1. Add 3mL LB/Amp (50 μl/mL) in each tube
2. Pick up plate and use tip to scrape a single colony
3. Soak the colony inside the incubation tube
4. Put the tubes on the shaker of 37℃ and incubate overnight(16hr)

Conventional assembly

Digestion

1. 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}

1. Adding each reagent ddH2O10XbufferDNARE

(37℃ incubator) After 2 hr digestion, put all the samples to go electrophoresis.

Electrophoresis preparation

1. Agarose gel synthesis:

1% Agarose: 0.8g agarose +80 ml TAE buffer

1. Mix and heat in microwave oven until boiling for about 2 minutes. (If EtBr is necessary, it is added after this step)
2. The mixture is added into the gel plate to create one 40ml well and two 20ml well.
3. 20 minutes is required to form the gel.

Electrophoresis

1. DNA mixture is loaded add loading dye (1.5 ) is added into the eppendorf.
2. 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

1. Add SYBR dye (must be protected from light, packed in an aluminum foil)
2. Add 5 of 10000X SYBR Safe to TAE solution and swirl the container gently to mix. (SYBR safe stock is on 4∘C refrigerator)
3. Sock the gel in the container; ensure the gel is fully immersed during staining.
4. Incubate for 15~20 minutes (continuously agitate the gel on an orbital shaker at 50rpm)
5. ※If the signal is not strong enough, the gel can be stained longer or the staining solution has to be replaced.

Gel extraction

1. Cut the insert and vector from the gel with blade
2. Put into gel extraction column and centrifuge 13,000 rpm for 30 sec
3. Use the elution samples for DNA ligation

Ligation

1. vector 0.5 μl
2. insert 1 μl
3. ligase buffer 1.5 μl
4. ligase 0.5 μl
5. ddH2O 11.5 μl
6. 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

1. Competent cells (100ul) were mixed with plasmids (15ul)(1ul for biobrick kit extract) in eppendorfs and put on ice for 30 mins.
2. They were under 42oC heat shock for 90 seconds and put on ice for 3 mins.
3. Luria-Bertani (LB) broth (1000ul) was added to the eppendorfs.
4. Cells were incubated at 37oC for 20 mins.
5. They were centrifuged for 2 mins. The supernatant was dropped, and the remaining medium was pipetted to resuspend the cells.
6. Cells were spread on LB agar plates with ampicillin, incubated for 18 hours. (37^oC)

E.coli culture

1. Add 3mL LB/Amp (50 μl/mL) in each tube
2. Pick up plates and use tip to scrape a single colony
3. Soak the colony inside the incubation tube (each plasmids 5 tube)
4. Put the tubes on the shaker of 37℃ incubate, overnight(16hr)

Selection

1. Spin down 1.0ml of E. coli overnight cells in a microcentrifuge. (13,000 rpm, room temp., 30~45 sec) and remove medium.
2. Add 50μl of TEN (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 100mM NaCl)
3. Vortex for 2 min. Turn upside down for 5 times. (VWR multivirtexer)
4. Add 70μl phenol/chloroform/isoamyl alcohol (25/24/1, with 1% β-ME)
5. Vortex for 2~3 seconds, microcentrifuge for 10 min.
6. Transfer upper solution 70 μl (without touching the interface) to new microcentrifuge tube.
7. Add 20μl of NH4OAc (7.5M) and 100μl of isopropanol (room temp.)
8. Vortex for 5 sec, microcentrifuge for 1 min (room temp.)
9. Wash the pellet with 70% EtOH(1ml) and dry it completely.
10. Use a paper towel to help dry the alcohol.
11. Re-suspend the pellet in 15μl of ddH2O and 1/50 RNase.
12. Add the ddH2O an d RNase together before adding into the eppendorf.

Digestion (check)

1. samples for each of 7 kinds DNA
2. Enzyme: EcoR I, Pst I, add 0.2 microliter to each reaction(15 microliter)
3. DNA: 5 microliter (3 if already done minipreparation)
4. Add 10X buffer, ddH2O, enzymes, DNA together
5. Digest in 37 degree incubator for 1.5 hrs

3A Assembly

Restriction Digest

Materials

1. Your two part samples: Miniprepped DNA (in BioBrick RFC[10] plasmid backbones)
2. Linearized plasmid backbone (with a different resistance to the plasmid backbones containing your part samples)
3. EcoRI, XbaI, SpeI, PstI
4. NEB Buffer 2
5. BSA
6. dH20

Procedure

Digest Reaction

1. dH20
2. NEB Buffer 2
3. BSA
4. DNA and Enzymes
5. Digest Part A with EcoRI and SpeI
6. Digest Part B with XbaI and PstI
7. Digest linearized plasmid backbone with EcoRI and PstI
8. Combine 1 ul of each restriction digest reaction with 1 ul of ligase in a 25 ul reaction.
9. Transform the ligation product.
10. If the input parts are good, almost all colonies will be correct.
11. If desired analyze the transformation with single colony PCR followed by agarose gel electrophoresis.
12. Miniprep clones that generated a band of the appropriate size.
13. Sequence the clone.
14. Record the sequence information in the Registry.

DNA Minpreparation

Tips for 10 min plasmid DNA minipreparation

1. Grow 2.0 ml overnight culture
2. Spin down 1.0 ml if E. coli overnight cells in a microcentrifuge (13,000 rppm, room temp., 30-45 sec) and remove medium.
3. Add 50 ul of TEN (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 100mM NaCl)
4. Vortex for 2 min. (VWR multivortexer)
5. Add 70 ul phenol/chloroform/isoamyl alcohol (25/24/1, with 1% β-ME)
6. Vortex for 2-3 sec, microcentrifuge for 10 min (room temp.)
7. Transfer upper solution 70 ul (without touching the interface) to new microcentrifuge tube
8. Add 20 ul of NH₄OAc (7.5M) and 100 ul of isopropanol (room temp.)
9. Vortex for 5 sec, microcentrifuge for 1 min (room temp.)
10. Wash the pellet with 70 % EtOH (1 ml) and dry it completely.
11. 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.