1,Template: plasmid OsNramp5-pFL61
2,Error-prone PCR Reaction System:Take 50 μL as an example
| Template Plasmid | 1-10ng |
|---|---|
| 2xStarMut Random System | 25ul |
| StarMut Enhancer | 10ul |
| Primer | 10μm each |
| ddH2O | make up to 50μl |
3,PCR program:Length of gene is about 1650bp
a.pre-denaturation: 95℃ for 2min.
b.denaturation: 95℃ for 30s.
c.Annealing: 63℃ for 1min.
d.Extension: 72℃ for 2 min. Repeat steps b to d for 25 cycles.
e.Complete extension: 72℃ for 7min.
f.Preservation: 4℃ for 10min.
4,Take 1-5 uL product and electrophorese it to detect strip concentration and specificity.
5,Take rest PCR product to electrophorese it, and recycle.
Ⅰ Plasmid Linearization
1,Primers: pFL61-HR-R and pFL61-HR-L . The primer concentration is 10uM.
2,System: taking 50uL as an example,
2x Phanta Max Master Mix 25uL
Primer each 2uL(pFL61-HR-R and pFL61-HR-L)
Template x uL （1ng）
ddH2O to 50μl
3,PCR: length of fragment is around 5000bp
a.pre-denaturation: 95℃ for 3min.
b.denaturation: 95℃ for 15s.
c.Annealing: 58℃ for 15s.
d.Extension: 72℃ for 5min. Repeat steps b to d for 30 cycles.
e.Complete extension: 72℃ for 10min.
f.Preservation: 4℃ for 10min.
4,PCR products were digested with DpnI to reduce the positive rate of circular plasmid template residues to clones. And we tried to precisely estimate the concentration of linearized plasmid by gel electrophoresis and purification
Ⅱ Homologous Recombination
1,Calculation
Multi-fragment homologous recombination reaction:
Most suitable dosage:
Clone vector =[0.02 × 5000] =100ng(0.03 pmol)
Each fragment=[0.04 × 1650]=66 ng(0.06 pmol)
2,Recombinant Reaction
(1)Amount of DNA according to the calculation To make sure the accuracy of sample,linearized carrier and insertion fragment shoul be diluted properly, and volume of each component should be more than 1μl.
(2)Formulation process of the following reaction system is on the ice box:
| Linearilized vector | Xμl |
| Insertion fragment | Yμl |
| 5xCEⅡ Buffer | 4μl |
| Exnase Ⅱ | 2μl |
| ddH2O | make up to 20μl |
1,Gently mix by the pipette(instead of oscillating mix), and collect the reacted mixture after short-time centrifuge.
2,Let it there stand still at 37℃，30min; cool it down to 4℃ or lay it on the ice.
1,Thaw cloning sensitive cell on the ice box;
2,Take 10μl recombinant product into 100μl sensitive cell liquid,flicking the tube wall to mix up(mustn’t oscillate to mix), and put it on the ice for 30min;
3,Water heating (42℃） for 45s，and put it on the ice to cool down for 2-3min;
4,Take 900μl LB culture without antibiotic,and sway it at 37℃ for 1h（200rpm);
5,Take LB solid culture with respective resistance to the 37℃ culture box to preheat;
6,Centrifuge at 5,000rpm for 5min, and abandon 900μl supernatant.Use the rest culture to resuspend the bacterium(E.coli) and spread the bacterium on the plate with right resistance with sterile spreading rod;
7,Invert to breed in the incubator at 30℃ for about 14h;
8,Add LB culture with respective resistance antibiotic into the test tube. Pick up cells that has been transformed into it. Sway it at 200rpm at 37℃ to mix it up.
1,Add 1-5ml bacterial fluid that has been bred for 12-16h into the centrifuge tube. Centrifuge at 10000xg for 1min. Abandon supernatant, turn it up and down on the absorbent paper to clean the rest fluid;
2,Add 250μl Buffer P1 into the centrifuge tube with bacterium precipitation(Please check Buffer P1 if it has been added RNaseA), and blow to mix it up;
(Notice:resuspending the bacterium throughly is very important for the productivity. After the resuspension, you wouldn’t see large block of bacterium.If there is no thorough mixed bacteria, it will affect lysis, resulting in low extraction and purity.)
3,Add 250μl Buffer P2 into the centrifuge tube. Turn it up side down slightly 8-10times, to make the bacterium(E.coli) split up completely. (within 5 min);
(Notice:Mix. The vortex causes genomic DNA break, resulting in mixing genomic DNA fragments in the extracted plasmid. At this time, the solution becomes viscous and translucent, indicating that the bacteria has been resolved. The time taken should not exceed 5 min to prevent the plasmid to be destroyed. If the solution is not clear, it may be appropriate to reduce the amount of bacterial amounts due to excessive cleavage caused by excessive bacteria.)
4,Add 350μl Buffer P3 into the centrifuge tube in the step3, inverting it 8-10 times slightly, to neutralize Buffer P2 completely. Then, there is white floc in appearing in the tube. Centrifuge at 13000×g for 10min;
(Notice:Invert the tube immediately after adding Buffer P3, to prevent local precipitation from affecting the neutralization. If there is any tiny white precipitation in the supernatant, take the supernatant after centrifuge second time)
5,Put FastPure DNA Mini Columns into Collection Tube 2ml. Transfer the supernatant in step4 into collection columns with pipette carefully,and mustn’t take in the precipitation. Centrifuge at 13000×g for 30-60sec. Throw away the liquid in the tube,and put back the collection columns into the collection tube ;
6,Add 500μl Buffer PW1 into the collection columns.Centrifuge at 13000×g for 30-60sec. Throw out the liquid,and put back the he collection columns into the collection tube ;
7,Add 500μl Buffer PW2(Please check if it is diluted with ethanol absolute) into the collection columns. Centrifuge at 13000×g for 30-60sec.Throw out the liquid, and put back the he collection columns into the collection tube ;
8,Repeat step8
9,Put back the he collection columns into the collection tube. Centrifuge collection column at 13000×g to dry it. Lay the open collection columns at a room temperature or in a warm box at 50℃, to clean the rinse liquid left in the column throughly.
(Notice: the ethanol in the rinse liquid has an impact on the following reaction, such as enzyme cutting,enzyme linking, PCR. This step can be neglected)
10,Put the collection column into a new sterile 1.5ml centrifuge tube.Add 30-100μL Elution Buffer into the collect column until it reaches the center of membrane.Lay it at room temperature for 2min, and then centrifuge at 13000×g for 1min to elute DNA. Elution Buffer need to be water bath before;
(Notice:The eluent volume should be more than 30μl, if it is less than 30μl，efficiency of elution will drop. If you need to get the highest yield, you warm the Elution Buffer to 55 ℃to increase the elution efficiency. Further, the solution obtained by centrifugation can be re-added into the centrifugation column, and then repeat step 10 . If subsequent sequencing is required to elute, and ensure that the pH of ddH2O is in the range of 7.0-8.5, the pH is lower than 7.0 reduces elution efficiency.)
11,Throw the collection column. DNA product should be preserved at -20℃，to prevent DNA from degrading.
1,Material:15ml centrifuge tube,50% PEG （4g PEG3350， 4g H2O）,transformation buffer,plate buffer
(1)Transformation buffer(10ml):
| 10×TE | 1mL |
| 10×LiAc | 1mL |
| dd H2O | 8ml |
(2)Plate buffer(1ml):
| 10×TE | 100μL |
| 10×LiAc | 100μL |
| 50%PEG | 800μL |
(50%PEG needs to be thawed by water bath at 55℃)
(3)Salmon testes DNA(10mg/ml) should be ready before the experiment.42℃ in the water bath kettle,boiled for 5 min, and cool it down to 4℃ in frige.
(4)10×TE(pH=7.5,100ml)
| 0.1M Tris-Hcl | 10mM EDTA |
| Tris-Hcl | 1.211g/100mL |
| EDTA | 0.29g/100mL |
Adjust pH to 7.5 with hydrochloric acid.20min sterilization treatment.
(5)10×LiAc（pH 7.5）（100mL）
1M LiAc 10.202g/100mL
Adjust pH to 7.5 with acetic acid.20min sterilization treatment.
2,Transformation
(1)Add 0.5mL transformation buffer （10×TE 1mL，10×LiAc 1mL）to 8ml ddH2O, and transfer it to 1.5ml centrifuge tube;
(2)Pick fresh yeast spots to 500μL transformation buffer. (Yeasts should be bred before)
(3)Remove supernatant after centrifuge at 5000g for 5min.Leave 100μl in the tube ,waiting it to suspend.
(4)Add 5μL 10mg/mL salmon test DNA(boiled for 5 min before we use).
(5)Add 5μl plasmid and mix.
(6)Add 600μL plate buffer 100μL 10×TE，100μL 10×LiAc，800μL 50%PEG）and mix.
(7)Constant temperature breeding at 30℃ for 4~16h.
(8)Heat it in the water bath kettle for 15min, mix it every 5min.
(9)Remove supernatant throughly after 5000g/5min centrifuge. Add 500μl ddH2O. Spread SD-ura plate with proper amount of mixture(100μl，or 50μl diluted）
1,SD-ura(100ml):
| -Ura | 0.077g |
| YNB | 0.67g |
| Glucose | 2%(w/v) |
Use NaOH to adjust pH to 5.7
Solid medium needs to be added into 2% agar
Add 450μL30μM CdCl2·5H2O（aq） into 150ml solid medium in liquid state before it cooling down,and shake well.
2,After determining the positive clones,add yeast liquid into the test tube with SD-ura liquid medium of 3~4ml.Sway the tube overnight at 220rpm at 30℃ overnight;
3,Take 200μl yeast liquid to detect absorbance. OD600 should be within 0.6~0.8.(adjust according to the growth of yeast);
4,Add 2ml yeast liquid in the centrifuge tube.Centrifuge at 5000g for 5min. Throw out the supernatant at the ultra-clean workbench;
5,Add 700μl ddH2O to wash . Centrifuge at 5000g for 5min after oscillation. Throw out the supernatant;
6,Add ddH2O according to OD(1:1),for example, OD=0.8+800μl ddH2O;
7,Take 100μl to have 10,100,1000 times dilution.(Spotting needs four levels, it has 10,100,1000,10000 dilution): Take 4 2ml centrifuge tubes, and add 900μl H2O to no.2,3,4 tube. Take 100μl liquid to the next tube to have a gradient dilution, starting from no.1 tube(centrifuge tube in the step5);
8,Spot.Take 10μl liquid, and drop on the SD-ura plate with final concentration of 30μM.
1,Take 2ml yeast culture liquid.Centrifuge at 12000rpm for 1min. Remove supernatant .
2,The disruption of yeast cell wall
Glass ball:Add 250μl LYP1 in yeast liquid(check if RNase has been added into it), fully suspending sediment.Add 200μL glass balls washed by acid, vortex oscillating for 10min. Centrifuge for a short time to sink it to bottom of tube, and take the supernatant to another clean centrifuge tube.
3,Add 250μl YP2 into centrifuge tube, and turn it upside down 6-8 times slightly to split yeast completely.
4,Add 350μl YP3 into centrifuge tube, and turn it upside down 6-8 times slightly, and shake well. There is white flocculent precipitation appearing at this time. Centrifuge at 12000rpm for 10min, and take the supernatant to another clean centrifuge tube with pipettor(try not to absorb the sediment).
5,Add supernatant we get in step4 into adsorption column.Lay it at room temperature for 2min.Centrifuge at 12000rpm for 1min. Drop the waste fluid and put absorption column back to collection tube.
6,Add 600μl washing into absorption column .Centrifuge at 12000rpm for 1min. Drop the waste fluid.Put absorption column back into collection tube.
7,Add 600μl washing into absorption column .Centrifuge at 12000rpm for 1min. Drop the waste fluid.Put absorption column back into collection tube.
8,Centrifuge at 12000rpm for 2min.Lay absorption open at room temperature or incubator for several minutes , to clean the residual washings. Otherwise, ethyl alcohol will influence the following experiment, such as enzyme cutting、PCR et al.
9,Put absorption column into a clean centrifuge.Drop 50-200μl eluant to the center of adsorption membrane . Lay it at room temperature for 2min.Centrifuge at 1200rpm for 1min.
10,To increase the recycling efficiency of plasmid, add eluant we get back into absorption tube. Lay it at room temperature for 2min.Centrifuge at 12000 rpm for 1min
1,+Cd SD-ura(100ml)
| -Ura | 0.077g |
| YNB(-Mn2+) | 0.67g |
| Glucose | 2%(w/v) |
| EGTA | 2.2821g |
Use NaOH to adjust pH to 6.0
Solid medium needs to be added into 2% agar
1.After determining the positive clones, add yeast liquid into the test tube with SD-ura liquid medium of 3~4ml. Sway the tube overnight at 220rpm at 30℃ overnight;
2.Take 200μl bacterium liquid to detect absorbance. OD600 should be within 0.6~0.8.(adjust according to the growth of bacterium);
3.Add 2ml bacterium liquid in the centrifuge tube. Centrifuge at 5000g for 5min.Throw out the supernatant at the ultra-clean workbench;
4.Add 700μl ddH2O to wash . Centrifuge at 5000g for 5min after oscillation.Throw out the supernatant;
5.Add ddH2O according to OD(1:1),for example,OD=0.8+800μl ddH2O;
6.Take 100μl to have 10,100,1000 times dilution. (Spotting needs four levels,it has 10,100,1000,10000 dilution):
Take 4 2ml centrifuge tubes, and add 900μl H2O to no.2,3,4 tube.Take 100μl liquid to the next tube to have a gradient dilution, starting from no.1 tube(centrifuge tube in the step5);
7,Spot. Take 10μl liquid,and drop on the SD-ura plate(absence of Mn2+).
