(→Protocol#5 : PCR clean-up and gel extraction) |
|||
| Line 445: | Line 445: | ||
|- | |- | ||
| Bind DNA | | Bind DNA | ||
| − | | Spin at 11,000g 30 s | + | | rowspan="2"|Spin at 11,000g 30 s |
|- | |- | ||
| Wash silica membrane | | Wash silica membrane | ||
Note : Everything should be done in sterile conditions and on ice (4°C). For 100ml of competent cells
For 100 mL of culture:
| KAc 1M | 1.5 mL |
| MnCl2 0.5M | 5 mL |
| KCl 1 M | 5 mL |
| CaCl2 0.1M | 5 mL |
| Gly 80% | 0.93 mL |
| H2O | 32.56 mL |
| NaMOPS 0.2M | 400 µL |
| CaCl2 0.1M | 6 mL |
| KCl 1 M | 8 mL |
| Gly 80% | 1.5 mL |
| KCl 1M | 80 µL |
| H2O | 500 µL |
To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL
| 25µL reaction | 50µL reaction | 100µL reaction | Final concentration | |
| EconoTaq PLUS 2X Master Mix |
12.5µL | 25µL | 50µL | 1X |
| Forward Primer (100 pmol/μL) |
0.5µL | 0.5µL | 1µL | 1 pmol/μL (1 μM) |
| Reverse Primer (100 pmol/μL) |
0.5µL | 0.5µL | 1µL | 1 pmol/μL (1 μM) |
| DNA Template* | 0.5µL | 1µL | 1µL | ? |
| Water nuclease-free |
[ ] μL qsp 25μL | [ ] μL qsp 50µL | [ ] μL qsp 100μL |
Cycling steps
| Cycling step | Temperature | Time | Number of cycles |
| Initial denaturation | 98°C | 3 min | 1 |
| Denaturation | 95°C | 10 sec | X27 |
| Annealing | 50-65°C | 40 sec | |
| Extension | 72°C | 1 min/kb | |
| Final extension | 72°C | 1 min/kb | 1 |
Preparation of 0.8% agarose gel
Sample preparation and migration
Revelation
Protocol used for a Macherey-Nagel purification kit, for more details see [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS.pdf here]. For better results use gloves and if so do not use gloves.
| Step | Actions |
| Cultivate and harvest cells | Centrifuge at 11,000g for 30 sec |
| Cell lysis | Add 250µL of buffer A1 Add 250µL of buffer A2 Incubate at room temperature up to 5 min 300µL buffer A3 |
| Clarification of the lysate | Centrifuge at 11,000g for 5 min |
| Bind DNA | Place a purification column in a collection tube (2mL) and carefully decant supernatant from the previous step, to a maximum of 750µL Then centrifuge at 11,000g for 1 min and discard the flow-through. Put the column back in the collection tube Repeat this step to load the rest of the supernatant if need be |
| Wash silica membrane | Add 600µl of buffer A4 Centrifuge at 11,000g for 1 min |
| Dry silica membrane | Centrifuge at 11,000g for 2 min |
| Elute DNA | Place the columns in an 1.5mL eppendorf and add 50 µL of buffer AE and incubate at room temperature for 1 min Centrifuge at 11,000g for 1 min |
To measure your DNA concentration, use a Biospec Nano apparel or Nanodrop apparel. Make the blank with 1µL of pure water and use 1µl of sample. Make sure to clear the lens with water and with a special paper in order to no line the lens.
Resuspend gBlocks Gene fragment to a final concentration of 10 ng/µL in TE or AE (elution buffer). The tube contain 1000 ng of gBlocks Gene fragment so you have to add 100µL of AE Mix well, a vortex shall be used
1. Digest 100 ng in 50µL
| component | 50 µL of reaction |
| DNA | 10 µL |
| Buffer 2 (10X) | 5 µL |
| BSA (100X) | 0.5 µL |
| H2O | 33 µL |
| EcoRI | 1 µL |
| PstI | 1 µL |
2. Incubate for 45 min at 37°C 3. Incubate 20 min at 80°C (inactivation of restriction enzyme)
1. Ligate 50 ng vector with a 3X molar excess of gBlocks Gene fragments in fresh T4 DNA ligase buffer diluted to 1X and 400 U T4 DNA ligase for a total volume of 20 µL
| component | 20 µL of reaction |
| PSBIC3 (or other vector) at 12.5 ng/µL | 2 µL |
| insert digested (at 37.5 ng/µL) | 12 µL |
| T4 Buffer | 2 µL |
| T4 ligase 400 U | 1 µL |
| H2O | 3 µL |
2. Incubate 2h at room temperature
To make negative control, follow the same procedure but without add plasmids and spread 300 µL
| DNA | Between 50 and 100 ng |
| EcoRI-HF | 0.2 µL |
| PstI | 0.2 µL |
| 10X NEBuffer 2 | 2 µL |
| H2O | QS 20 µL |
Incubate all digest reactions at 37°C for 1 hour and then add 3 µL of SES 4X and migrate 30 min at 150V on a 1% agarose gel.
| Upstream part plasmid | 500 ng |
| EcoRI-HF | 1 µL |
| SpeI | 1 µL |
| 10X NEBuffer 2 | 5 µL |
| H2O | To 50 µL |
| Downstream part plasmid | 500 ng |
| XbaI | 1 µL |
| PstI | 1 µL |
| 10X NEBuffer 2 | 5 µL |
| H2O | To 50 µL |
| Destination plasmid | 500 ng |
| EcoRI-HF | 1 µL |
| PstI | 1 µL |
| DpnI | 1 µL |
| 10X NEBuffer 2 | 5 µL |
| 100X BSA | 0.5 µL |
| H2O | To 50 µL |
Incubate the three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
| Upstream part digestion | 2 µL |
| Downstream part digestion | 2 µL |
| Destination plasmid digestion | 2 µL |
| 10X T4 DNA ligase buffer | 2 µL |
| T4 DNA ligase | 1 µL |
| H2O | 11 µL |
Incubate at RT for 1 hour.
| PCR clean-up | Gel extraction | |
| PCR clean-up, DNA clean-up, or single stranded DNA clean-up: Adjust binding conditionGel extraction: Excise DNA fragment / solubilize gel slice | 200 μL NTI/ 100 μL PCR | 200 μL NTI/ 100 mg gel 50 °C 5–10 min |
| Bind DNA | Spin at 11,000g 30 s | |
| Wash silica membrane | 700 μL NT3 11,000g 30 s Recommended: 2nd wash 700μL NT3 11,000g 30 s |
From a overnight starter, (3mL in 2YT medium + appropriated antibiotiques) from the strand holding the transduct mutation, make a 1/100 dilution in 3mL 2YT medium and add 150µL of Cacl2 0.1M ( without antibiotique corresponding of the transduct allele). Incubate at 37°C (under agitation) until obtening a DO600=0.5-1 (more 0.5) Add 150µL of a P1 lysat wt Incubate at 37°C without agitation (water bath) during 20min Incubate at 37°C under agitation during 3 or 4h (even overnight) Divide the culture in 2 eppendorfs of 2mL (=2*1.5mL) Add 150µL chloroform then vortex Centrifuge 10min at 4°C and 6000rpm Transfer 1mL supernatant into 2 news eppendorf of 2mL Add 10µL chloroform (under a fumehood) in each eppendorf, vortex and keep at 4°C (option: to increase the titration level, you can do a second cycle)
The eve, prepare a culture of the recipient strain in 3mL 2YT medium (+eventual appropriated antibiotique) The D-day, start a culture from the recipient strain (1/50 dilution) in 3mL 2YT + 150µL of CaCl2 0.1M ( prepare the LB-citrate 5mM requiered and LBagar- appropriated AB + citrate 2mM petri dishes) Incubate until reaching a DO600 = 1 or + (2.10^6 bact/mL) Prepare 3 tubes eppendorf of 2mL (1 control with 500µL cells, 1 with 10µL P1 + 500µL cells, 1 with 100µL P1 + 500µL cells) Infect during 20min at 37°C (heat wuthout agitation) Add 1mL steril LB-citrate 5mM and heat at 37°C during 50min under agitation (700-750rpm) Centrifuge 5min at 5000rpm (RT) Remove supernatant sterilely with a P1000 and resuspend in 1mL steril LB-citrate 5mM Spread on LBagar- appropried AB + 2mM citrate Incubate ON at 37°C
Isolate clones on petri dishes (approprieted AB + citrate 2mM) Incubate at 37°C
Test clones by PCR Transplant the good clones on LB-AB appropriate petri dishes
The assays were performed in a total volume of 500µL, containing 500mM sodium acetate buffer (ph = 6.0), 10mM L-lysine, 0.1 mM pyridoxal-5-phosphate, and 20µL whole cells, at 37°C in a water bath. The reaction was stopped after 24h by the addition of 10µL ethanol. The reaction mixtures were centrifuged and the 30µL of supernatant was applied to derivatization to determine the amounts of residual lysine and product, i.e cadaverine.
Diamine deritatives were obtained by the reaction of 180µL of borate buffer 0.05M (pH9), 60µL of 100% methanol, 47µL diethyl ethoxymethylenemalonate without any pretreatment. The samples were heated at 70)C for 2h to allow complete degradation of excess DEEMM and derivatization.
After derivatization with diethyl ethoxymethylmalonate, analyses were performed on a high performance liquid chromatograph (HPLC Agilent technologies, 1260 Infinity) consisting of a binary pump, an inline degasser, an autosampler and a column thermostat. Chromatographic separation was carried out by reverse-phasechromatography on a C18 column maintained at 35°C. Mobile phase A was composed of 100% acetonitrile, and B was made up of 25mM aqueous sodium acetate buffer (pH = 4.8). The flow rate of 1mL/min was used, with the following gradient program : 0-2min, (20-25% A; 2-32min, 25-60%A; 32-40min, 60-20%A. Detection was carried out at 284nm.
1. Digest vector with restriction enzyme during 3h and purify the linearized vector with a commercial PCR purification kit. Elute DNA with elution buffer or 10mM TrisCl, pH 8.0-8.5. Do nnot elute the DNA with water or TE. Measure the concentration of the vector.
2. Amplify your gene of interest by PCR using primers with 15 mer (or inferior) homology extension to the linearized vector end. We usually use 15bp homology for single fragment cloning, and 20bp for multiple fragment cloning. Purify the linearized vector with a commercial PCR purification kit. Elute the DNA with water or TE. Measure the concentration of the insert(s).
3. Mix the linearized vector and insert at a molar ratio of 1.2 ina 1.5mL tube. An examble is shown as follows. (Vector to insert molar ratio of 1/1 for single fragment cloning, 1:2:2 for multiple fragments cloning. AN example of 3 fragments cloning is shown below with vector: insert 1: insert 2 molar ratio as shown is 1:2:2 as shown in Fig. 4A).
| Stock concentration | Volume added | Final concentration | |
| Linearized vector | 100ng/µL | 1µL | 10ng/µL |
| Insert 1 | 40ng/µL | 1µL | 4ng/µL |
| Insert 2 | 40ng/µL | 1µL | 4ng/µL |
| 10X BSA | 1µL | 1X | |
| 10X NEB Buffer 2 | 1µL | 1X | |
| H2O | Up to 10µL |
4. Add 0.2µL of T4 DNA polymerase (3U/µL, NEB) to the mixture and incubate 2min at room temperature
Soft geloses have been made by adding 0.3% agar in LB medium. Once dried, each bacteria strain has been spotted using a tooth pic on the petri dishes and incubated for 3h in a 37°C room.
| DesA slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGCGCTCGCACTTGCTT |
| desA slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGAGGCACGGTC |
| desB slic forward | "a rentrer" |
| desB slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTACACTGCAAATTC |
| desC slic forward | "a rentrer" |
| desC slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGGCTGAAACCGC |
| desD slic forward | "a rentrer" |
| des D slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACGCCCGGCTAA |
| FliC E. coli slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT |
| FliC E.coli slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG |
| FliC desulfo slic forward | cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGTCACTGGTTATCAAT |
| FliC desulfo slic reverse | ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAGCCGCCGAGAAG |
| CsgA E. Coli slic forward | "a rentrer" |
| CsgA E. Coli slic reverse | "A rentrer" |
