Team:SCUT China/Notebook
Our team have seven wet lab experimenters, all of whom are laboratory rookies at first. We still remembered how hard for us to spend about 3 months completing the first plasmid construction. After one year iGEM training, we are skilled in molecular cloning operation - can complete multiple plasmids construction simultaneously in 3 days at the soonest now. During this time, many experiments failures we have experienced. But we have got a lot of joy from the success of the experiments at the same time. Through the training for the whole project, we deeply understand the importance of laboratory safety and strict implementation of protocols when performing an experiment. Consequently, we would like to post our protocols detailed enough for other team to repeat our experiments. Simply click on a protocol to read its steps. The side menu to scroll to the specific protocol is convenient for you to look for.
(1)Lysogeny Broth (LB)
Formula:
NaCl 1%
Tryptone 1%
Yeast extract 0.5%
Agar 2% (Solid)
(2)Antibiotic-concentrations:
Antibiotic |
Formula |
Standard Concentration |
Working Concentration |
Ampicillin |
1g+10mL ddH2O |
100mg/mL |
25-50μg/mL |
kanamycin |
0.1g+10mL ddH2O |
10mg/mL |
10-50μg/mL |
Chloramphenicol |
0.25g+10mL ethanol |
100mg/mL |
2.5-25μg/mL |
Carbenicillin |
1g+10mL ddH2O |
100mg/mL |
25-50μg/mL |
Tetracycline |
0.1g+10mL ddH2O |
10mg/mL |
10-50μg/mL |
(3)Filtration Sterilization
Acrodisc 32mm syringe filters with 0.2μm Supor membrane are used to remove bacteria from liquids.
(4)Culture in Incubator&in Shaker
In Incubator: at 37℃
In Shaker: at 37℃ and 220rpm
(5)Glycerol Stock
<1> Add 250μl 60% glycerol to 750μl bacteria solution and mix them thoroughly.
<2> Store at -80℃.
Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume.
(1) Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).
(2) Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15- 25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)
(3) Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
Note: No cell clumps should be visible after resuspension of the pellet, otherwise incomplete lysis will lower yield and purity.
(4) Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times. Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
(5) Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.
(6) Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
(7) (Optional) Wash the Spin Column CP3 by adding 500 µl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and put Spin Column CP3 back to the collection tube. This step is recommended to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.
(8) Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.
(9) Repeat Step 8.
(10) Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.
Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.
(11) Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Buffer EB to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid recovery efficiency.
Ensure that Buffer GD and Buffer PW have been prepared with appropriate volume of ethanol (96-100%) as indicated on the bottle and shake thoroughly.
(1) Pipet 1-5 ml bacterial culture suspension in a centrifuge tube, centrifuging for 1 min at 10,000 rpm (~11,500 × g), discard supernatant as possible.
(2) Add 200 μl Buffer GA. Mix thoroughly by vortex.
Note: For difficult-broken Gram-positive bacteria, Step 2 could be replaced by lysozyme treatment: add 180 μl enzymatic lysis buffer (20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA; 1.2% Triton® X-100; lysozyme (final concentration of 20 mg/ml)). Incubate for at least 30 min at 37°C. Lysozyme should be prepared with buffer, otherwise the lysozyme is not active. If RNA-Free genomic DNA is required, add 4 μl RNase A (100 mg/ml, should be prepared by user, Cat. no. RT405-12), mix by vortex for 15 s, and incubate for 5 min at room temperature (15- 25°C).
(3) Add 20 μl Proteinase K. Mix thoroughly by vortex.
(4) Add 220 μl Buffer GB to the sample, vortex for 15 s, and incubate at 70°C for 10 min to yield a homogeneous solution. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid.
Note: White precipitates may form when Buffer GB is added. They will not interfere with the procedure and will dissolve during the heat incubation at 70°C. If precipitates do not dissolve during heat incubation, it indicates that the cell is not completely lysed and may result in low yield of DNA and impurity in DNA.
(5) Add 220 μl ethanol (96-100%) to the sample, and mix thoroughly by vortex for 15 s. A white precipitate may form on addition of ethanol. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid.
(6) Pipet the mixture from step 5 into the Spin Column CB3 (in a 2 ml collection tube) and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard flow-through and place the spin column into the collection tube.
(7) Add 500 μl Buffer GD (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s, then discard the flow-through and place the spin column into the collection tube.
(8) Add 600 μl Buffer PW (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard the flow-through and place the spin column into the collection tube.
(9) Repeat Step 8.
(10) Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to dry the membrane completely. Note: The residual ethanol of buffer PW may affect downstream application.
(11) Place the Spin Column CB3 in a new clean 1.5 ml centrifuge tube, and pipet 50-200 μl Buffer TE or distilled water directly to the center of the membrane. Incubate at room temperature (15-25°C) for 2-5 min, and then centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. What’s more, the pH value of eluted buffer will have some influence in eluting, we suggest chose buffer TE or distilled water (pH 7.0-8.5) to elute gDNA. For longterm storage of DNA, eluting in Buffer TE and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis.
All Primers used in experiments meet basic requirements below:
- Free 3’-end.
- Number of Mismatches<150.
- 55℃<Tm<72℃
- 18 bp<Length<25 bp
- 40%<GC Content<60%
The Softwares Oligo7 and SnapGene are used to design primers.
Especially:
- For Gibson Assembly
- 68℃<Overhand Tm<72℃
- 45%<GC Content<55%
- The difference between Tm of overhand of each primer is no more than 2℃.
- The length of part combining with targeted fragment is between 18bp and 25bp.
- The difference between Tm of combining parts of two primers is no more than 5℃.
- For sequencing
<1> The length is around 20bp.
<2> The number of hairpin structures and primer dimers is less than 3 respectively. And the number of mismatches is strictly less than 100.
(3) For overlap PCR
55℃<Tm of each primer<60℃
Primers preparation: Carefully hold the lyophilized primer powder and centrifuge at 12000rmp for 2min. Dilute to 20 μM with ddH2O.
(1) Q5 PCR
10μl Assay |
Step |
Temperature (℃) |
Time |
Cycle |
|
5×T5 Reaction Buffer |
2 |
Initial Denaturation |
98 |
30 s |
1× |
dNTPs |
0.8 |
Denaturation |
98 |
10 s |
30× |
Forward primer |
0.25 |
Annealing |
|
30 s |
|
Reverse primer |
0.25 |
Elongation |
72 |
|
|
Template |
Dependent |
Final Elongation |
72 |
2 min |
1× |
Q5 polymerase |
0.1 |
Hold |
4 |
∞ |
1× |
ddH2O |
Dependent |
|
|
|
|
<1> The temperature of annealing=Tm of primers-5℃.
<2> The final concentration of each template is 1ng/μl.
<3> The time of elongation depends on the length of fragments to be amplified:
<1 kb: 15 s-30 s
≥1 kb: 40 s/kb
≥2.5 kb: 1 min/kb
(2) T5 PCR
10μl Assay |
Step |
Temperature (℃) |
Time |
Cycle |
|
2×T5 Super PCR Mix (Colony) |
5 |
Initial denaturation |
98 |
30s |
1× |
Forward primer |
0.2 |
Denaturation |
98 |
10s |
29× |
Feverse primer |
0.2 |
Annealing |
Tm-5 |
30s |
|
Template |
2 |
Elongation |
72 |
30s/kb |
|
ddH2O |
2.6 |
Final elongation |
72 |
2min |
1× |
|
|
Hold |
4 |
∞ |
1× |
(3) Overlap PCR
Step 1:
25μl Assay |
Step |
Temperature (℃) |
Time |
Cycle |
|
Buffer |
5 |
Initial Denaturation |
98 |
30s |
1× |
dNTPs |
2 |
Denaturation |
98 |
10s |
5× |
Template 1 |
Dependent |
Annealing |
Tm-5 |
30s |
|
Template 2 |
Dependent |
Elongation |
72 |
30s/kb |
|
Enzyme |
0.25 |
Final Elongation |
72 |
2min |
1× |
ddH2O |
|
Hold |
4 |
∞ |
1× |
*The final concentration of each template is 50ng/μl.
Step 2:
50μl Assay (μl) |
Step |
Temperature (℃) |
Time |
Cycle |
|
Buffer |
5 |
Initial Denaturation |
98 |
30s |
1× |
dNTPs |
2 |
Denaturation |
98 |
10s |
25× |
Forward primer |
1.25 |
Annealing |
Tm-5 |
30s |
|
Reverse primer |
1.25 |
Elongation |
72 |
30s/kb |
|
Template |
25 |
Final Elongation |
72 |
2min |
1× |
Enzyme |
0.25 |
Hold |
4 |
∞ |
1× |
ddH2O |
15.25 |
|
|
|
|
(4) Colony PCR
<1> Pick several colonies and transfer them respectively to different grids (drew outside the bottom of the dish) of a new culture plate.
<2> Culture the bacteria for 8-10h.
<3> Add 20μl ddH2O to PCR tubes.
<4> Scrape appropriate amounts of bacteria cells and solve them in ddH2O.
<5> Lyse the cells at 95℃ for 10min.
10μl Assay |
Step |
Temperature (℃) |
Time |
Cycle |
|
2×T5 Super PCR Mix (Colony) |
5 |
Initial denaturation |
98 |
30s |
1× |
Forward primer |
0.2 |
Denaturation |
98 |
10s |
29× |
Feverse primer |
0.2 |
Annealing |
Tm-5 |
30s |
|
Template |
2 |
Elongation |
72 |
30s/kb |
|
ddH2O |
2.6 |
Final Elongation |
72 |
2min |
1× |
|
|
Hold |
4 |
∞ |
1× |
(1) Preparation of 1% gel
Size |
Agarose (g) |
1×TAE buffer (ml) |
DNA stain (μl) |
Big |
0.8 |
80 |
2 |
Medium |
0.4 |
40 |
1 |
Small |
0.2 |
20 |
0.5 |
<1> Measure out appropriate amounts of agarose and add corresponding TAE buffer.
<2> Heat the solution until it becomes clear. Add appropriate amounts of DNA stain after it cools down to about 55℃.
<3> Pour the solution to the mold with a comb.
<4> Let it solidify for at least 20min.
(2) Move the comb. Put the gel with the mold in the electrophoresis chamber with TAE buffer.
(3) Add 1/10 10×Loading buffer to each DNA sample.
(4) Pipet samples and DNA marker to wells.
(5) Run for 20-30min at 120V.
(1) Gel Extraction
<1> Place the gel under the ultraviolet lamp and quickly cut off the gel containing the targeted DNA fragments. Remove excess gel as much as possible.
<2> Weigh the gel and put it in a 1.5 mL centrifuge tube. Add 100μL Buffer GDP every 100mg gel.
Water bathe the gels at 50-55℃ for 7-10 minutes to dissolve them completely. During the water bath, turn upside down the tube twice to fasten dissolution. If the length of DNA fragment is no more than 100bp, add 3 times volume of Buffer GDP, and equal volume of isopropanol after the water bath.
<3> Centrifuge briefly to collect droplets on the tube wall. Place a HiPure DNA Column in a 2mL collection tube. Transfer the solution (≤700μL) to the column. Centrifuge for 30-60 seconds at 12,000 × g. If the volume of solution exceeds 700μL, discard the filtrate and put the column back into the collection tube. Transfer the remaining gels to the column and centrifuge at 12000 × g for 30-60 seconds.
<4> Discard the filtrate and put the column back into the collection tube. Add 300 μL Buffer GDP to the column. Set it still for a minute. Centrifuge for 30-60 seconds at 12,000 × g.
<5> Discard the filtrate and put the column back into the collection tube. Add 600 μL Buffer DW2 (has been diluted with absolute ethyl alcohol) to the column. Centrifuge for 30-60 seconds at 12,000 × g.
<6> Repeat step <5>.
<7> Discard the filtrate and put the column back into the collection tube. Centrifuge at 12,000 x g for 2 min. Open the lid of the column and dry for 10-15 minutes in the air to remove ethanol thoroughly.
<10> Place the column into a clean 1.5mL centrifuge tube and add 7-30μL water (preheated to 55℃) to the center of the column membrane. Set it still for 2 minutes. Centrifuge at 12,000 × g for 1 min.
<11> Repeat step <10> to increase yield.
<12> Remove the column and store the DNA at -20℃.
(2) Column Purification
Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume). All centrifuge steps are in a conventional tabletop microcentrifuge at room temperature (15-25°C).
<1> Column equilibration: add 500 Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB1 back into the collection tube (please use freshly treated spin column).
<2> Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene.
Note: For example, add 250 μl Buffer PB to 50 μl PCR reaction (not including oil).
<3> Transfer the mixture to the Spin Column CB1, incubate at room temperature (15-25°C) for 2 min. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube.
Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.
<4> Add 600 µl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB1 and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and place Spin Column CB1 back in the same collection tube.
Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5 min after adding Buffer PW, and then centrifuge.
<5> Repeat step <4>.
<6> Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry
the membrane.
Note: Residual ethanol from Buffer PW may inhibit subsequent experiment (enzymatic or PCR reactions).
<7> Place the Spin Column CB1 in a clean 1.5 ml microcentrifuge tube. Add 20-50 μl Buffer EB to the center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 20 μl, it may affect recovery efficiency. The pH value of eluted buffer will have big influence in eluting; distilled water (pH 7.0-8.5, adjusted with
NaOH) is suggested to elute plasmid DNA, pH<7.0 will decrease elution efficiency. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in
water is subject to acid hydrolysis. Repeat step 7 to increase plasmid recovery efficiency.