Team:NJAU China/Experiments
Overview
n this year's competition, we still use the E. coli as our chassis. Therefore, it mainly involves the experimental theory of Microbial Biology and molecular biology. Before the start of the experiment, we carried out a detailed design of the experiment scheme, and carried out the pre-experiment. For the detailed experimental arrangement, please look at the contents of the page below.
Overall Arrangement
Reagent and Medium
ysogeny broth (LB): 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl with suitable antibiotics.LB agar plate: LB medium with 1.5% (w/v) agar.
- Kanamycin: 25 μg/mL
- Ampicillin: 50 μg/mL
- Chloramphenicol: 35 μg/mL
- Glycerol for storing strain: 50%
- Ethanol for sterilization: 75%
Competent Cells
e use strain DH5α (for common plasmid or cloning transformation)、BL21 and Hfr (for expression experiment transformation) to make competent cells.
The composition of TSB buffer (100ml):
- LB: 85ml (pH6.1)
- PEG3350：10g
- Glycerol: 10ml
- DMSO: 5ml
- MgCl2: 10mM
- MgSO4：10mM
Transformation
or plasmid transformation, we add 2-5µl DNA (about 100ng/µl) into 50µl competent cells. For linearized product transformation, we add 10µl DNA (about 50ng/µl) into 50µl competent cells. Usually, we will make a control transformation. The next procedure is as the following description. The mix of DNA and competent cells incubates on ice for 30min, then is heat shocked at 42°C for 1 min and incubate on ice for 5min. Pipette 500µl LB to each transformation and shake incubate for an hour at 37°C. Next, pipette each transformation on two petri plates for a about 50µl and 300µl plating.Incubate transformations overnight (12-16hr) at 37°C to observe the result. We need to pay attention that before adding LB into the tube, we should be carefully and gently.
A Colony PCR
his protocol is frequently used in our examination of colonies, which are from linearized product transformations. Just pick single colonies from transformations to do a colony PCR. If the results are right, do the further experiments.
Miniprep
e use GENERAY GK1002-200 miniprep kit for minipreps and do as their guidelines.
Plasmid Construction
or constructing plasmids, we have tried a lot of ways, including plasmid double restriction, plasmid single restriction, T-A colony, PCR product restriction, PCR overlap and 3A assembly. We use T4 ligase for ligations. But we have some difficulties in constructing large plasmids. The procedures are shown below. The enzymes are from TAKARA.
- Long DNA PCR (>5kb)
DNA polymerase: PrimerSTAR
DNA：x bp
Reaction System (25μl):dH2O 14.7(μl) 5XPS Buffer(Mg2+ plus) 5 dNTP Mixture 2 primer 1 1 primer 2 1 PrimeSTAR 0.3 DNA 1 Reaction Program (30cyc):
98℃ 98℃ 52℃ 72℃ 72℃ 15℃ 2min 30s 15s (x/1000+3)s 10min ∞ - Not very long DNA PCR
DNA polymerase: rTaq
DNA：x bp
Reaction System (25μl):dH2O 13.2(μl) 10x Buffer(-Mg2+) 5 dNTP Mixture 2 Mg2+ 2 primer 1 1 primer 2 1 rTaq 0.3 DNA 0.5 Reaction Program (30cyc):
94℃ 94℃ TM+-5℃ 72℃ 72℃ 15℃ 2min 30s 30s (x/1000+3)s 10min ∞ - T-A colony
Materials: PCR product adding A tail, pMD®18-T Vector,
System (10μl):pMD®18-T Vector*1 1μl DNA 1-3 μl dH2O Adding whole to 10μl Solution I 5μl
Gene Knock-out
e have tried using lambda recombination to knock out traI gene, but failed. Finally, the work is done by Genescript Company.