Team:SJTU-BioX-Shanghai/Contribution

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J23117 is a weak promoter and it can lead to a quite low expression level in the absence of dCas9-ω. So, it is a good choice to be designed as a part of CRISPRa system. I It will cause a significant difference in expression level of reporter after dCas9 binding.

Figure above: Quoted from a study by Broad Institute

W103 and W108 in the figure means different ways to put the target site for dCas9-ωupstream of the promoter. Comparing the dCas9-ω activation of GFP expression from the three constructs using different gRNA, J23117 performs best when induced by the dCas9-ω both in W103 and in W108.

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Troubleshooting

Every team may encounter various problems during the experiment. This year, we have made a troubleshooting manual based on our own experimental experience and lessons. This manual contains a number of problems that are very likely to appear in the experiment, and also contains the solutions to some of the problems. We hope that such a manual can help more iGEM teams and students.

Agarose gel electrophoresis

For all operations at the electrophoresis table, please make sure that you wear laboratory clothes and plastic gloves. Tie your hair when necessary.

Prepare agarose gel

1. Calculate the amount of agarose from the concentration and the total volume (each small piece of gel needs 25ml 1×TBE). Pay attention to the concentration of the gel (not all the gel concentration is 1%. The gel with low concentration (about 0.8%) is used to distinguish large fragments. And high concentrated(about 1.5-2.5%) gel can distinguish small fragments)
2. Remember to add dyes in addition to the agarose and TBE when preparing the gel.
3. Make sure the agarose is completely melted in TBE before pouring it (usually it needs to be heated and boiled twice in the microwave).
4. Set the baffle and comb of the gel tank in the electrophoresis tank and sel the baffle inside until the gel is cooled.
5. Make sure that the gel is cooled when you pull out the comb. If it is not cooled, do not plug the comb back in, which will cause the swim lane to distort.
6.If the gel will not be used within 2 hours, preserve gel in TBE in time after cooled for about half an hour; if you want to leave it overnight, put it in a plastic bag and preserve it in the refrigerator at 4℃.

Agarose gel electrophoresis

1. Make sure that the gel is cooled before used.
2. Pay attention to the orientation when putting the gel into the electrophoresis apparatu.
3. When running the gel for verification, calculate the system before adding the sample: DNA is about 50ng, add water to make up to 3μl (if it exceeds, then 5μl), add 1μl of Buffer; try to keep the same volume for each sample and marker to control variables.
4. Control the running time of the gel, not too long to prevent it from running out (watching the process if you are not sure), look at the purple strip for judgment.
5. When using the electrophoresis, confirm whether it has a constant voltage. If current cannot be adjusted, pay attention the corresponding voltage (3~5V per cm electric field length is appropriate).
6. When running two pieces of gel at the same time, be aware that the bands of the two pieces of gel may be quite different, sometimes you need to take out the smaller piece of gel first.
7. Do not let the gel soak in the electrophoresis box for too long after running the gel, which may cause the band to diffuse.
8. If there is bubble in the TBE, replace the TBE in the electrophoresis box in time.
9. To run the gel, put the gel straight, otherwise the strip will be distorted.
10. At the end, look at the gel chart and put the gel on the instrument pad to avoid air bubbles.

Select monoclones

1. When shaking the bacteria, pay more attention to the turbidity of the medium. We need to observe the tuibidity to speculate the bacteria's growing phase.
2. If you use a pipette to select monoclones, remember to disinfect the gun in advance (ultra-clean UV).
3. If you use tweezers to pick bacteria, remember to burn the tweezers on the flame again after each time you select monoclones, and wait for it to cool before proceeding to the next picking.
4. Under normal circumstances, do not puncture/scratch the culture medium when picking clones.
5. After picking the clone, The lid of the centrifuge tube must be covered, otherwise it will be easily loose the lid in the shaker and get contaminated.
6. The inoculation loop should be used for picking clones and coating plates. If it is divided into sections, remember to burn the inoculation loop after each scribing before proceeding to the next scribing.

PCR design

Primer design

1. Please try to use 22~35mer (KOD kit Tm value*>63℃) primers.
2. Please design primers with GC contents within 45~60%. Please also confirm the position of the GC (bias): If the GC is close to the 3'terminal, the dispersion and banding will easily occur. (It is ideal for the design to be 60~70% on the 5'side and 40~50% on the 3'side.)
3. The 3' terminal is designed as G or C residue to improve the efficiency of primer and template binding efficiency. and banding.
4. Please make sure that the primers do not form intramolecular secondary structure, primer dimers, etc.
5. When amplifying long fragments, please use primers with a length of 25~35mer and a Tm value of 65 ℃ or higher. The success rate of primers above 25mer (Tm value≥65℃) is higher.

PCR system setting

1. Better use premix! ! ! (Avoid pipette error)

2. Take a tube of ddH2O daily from the refrig aterator 4℃, and mark the opening date after use.
3. When adding samples, start from the largest volume, add template and enzyme at the end (add the enzyme last), try not to add to the wall of the tube, if you accidentally add to the wall, you can use a small centrifuge to centrifuge briefly.
4. dNTP and MgSO4 melt slowly, and can be taken out from refrigerator in advance and melts at room temperature.

DNA concentration and purity determination

1. Use ddH2O to rinse machine when opening the program for the first time.
2. After opening the program, blank with eluent (ddH2O or EB).
3. After measuring once, rinse the sample area with blank and wipe it clean after washing.
4. After the last test, remember to put the paper in between back.

Transform

1. The competent cells that have been thawed can not be freezed back to -80℃.
2. After adding DNA to competent cells, use pipette tip to stir gently.
3. Competent cells cannot be centrifuged with a too high speed (3000r-5min is appropriate).
4. The heat shock time and temperature of different competent cells maybe different, so special attention is required.
5. Post a note during the water bath to prevent others from changing the temperature.
6. The volume of plasmids added should not exceed 10% of competent cells.
7. Open the ultra-clean table in advance to avoid exceeding the specified time.
8. Pay special attention to the coating resistance.

Prepare culture medium

1. Make sure what kind of culture medium is needed, the solid or the liquid.
2. When adding the agar powder, be careful not to make the weighing paper wet.
3. If the sterilized solid culture medium is not going to be poured into the plate immediately, put it in the oven to prevent solidification.
4. When sterilizing the culture medium, seal the sterilization conical flask with two layers of tin foil to prevent contamination.

Prepare antibiotics

1. Find enough filter membrane before preparation.
2. Remember to sterilize the EP tubes in advance, and sterilized EP tubes cannot be opened outside. Open it directly in super clean beach.

Sterilization

1. Safety comes first!
2. The 15mL centrifuge tube and ddH2O bottle should not be screwed too tightly.
3. Before the sterilization starts, observe the water level of the sterilization pot.
4. Before the sterilization starts, make sure that the vent valve is closed.
5. Make sure that the closing light is off before clicking “work” button.
6. At the end of sterilization, before opening the sterilization pot, don't open the vent valve until you can't hear the "hissing" air leak . Ensure that the internal and external air pressures are equal.
7. After the EP tubes are sterilized, put the box containing EP tubes in the oven to dry them.

Digestion

1. Before digestion, pay attention to the number of bases in the cut fragment, and pay attention to adjusting the amount of template added in the digestion system.
2. Adjust the tempreture of the water bath in advance.
3. Remember to check the expiration date before using the enzyme (if the enzyme is found to be expired, give feedback in time).
4. 20ul for digestion and elution.

Enzyme Link

1. If the enzyme connection fails, you can try different ratios to explore the conditions.
2. It is recommended to use 0.5ul enzyme for one hour (20ul system), 22-23℃.

Gibson assembly

1. Matching system (the fragment should not be less than 200bp as far as possible).

2. Turn on the water bath in advance.
3. The template must be digested.
4. Assembly time 4h.

Extraction and purification

Plasmid

1. The protocol in the kit doesn't contain : before adding EB for elution, the elution column should be cooled for more than 15 minutes so that the alcohol will fully evaporate.
2. Preheat the EB/ddH2O before use (take a little more than the required amount of EB/ddH2O solution in the EP tube and preheat it in a 55℃ water bath).
3. EB/ddH2O is eluted twice (the first eluate is added back to the column and washed the second time).
4. It is better to wash small fragments with ddH2O (but ensure that the ethanol is completely evaporated), and ddH2O must be used for sequencing/electrotransmission.

Gel purification

1. Prepare some weighed EP tubes in advance.
2. When cutting the gel, put a plastic layer under the gel to avoid scratching the board below.
3. Before heating and melting, you can use a pipette tip to break the gel a little bit, and remember to blow the pipette (to blow out the remaining gel in the pipette tip) after poking, and then weigh the total mass.

Column purification

1. Cool the product for at least 20 min.
2. Elute with ddH2O.

other

1. Be careful not to expose the bacteria directly to the ultraviolet lamp of the ultra-clean bench.
2. If a large can of ddH2O after sterilization is to be opened, it should be used next to the alcohol lamp in super clean bench.
3. Please remember to write the date when naming any sample, and write down the detailed information of the product corresponding to the sample name in the experiment logbook.
4. Remember to turn off the power after using the balance.
5. Remember to adjust the volume after the pipette is used up.
6. When adding a small volume of liquid, be careful and make sure you add successfully.
7. After opening the shaker, make sure that the shaker works again before leaving.

Experiment record writing (paper + online)

1. Remember to add the gel map with the legend.
2. Remember to write experimenter.
3. Remember to date the samples.
4. Remember to write the detailed information of the sample in the corresponding sample tube (name + detailed information of the content + rough storage point).

Placed in order of date! ! ! ! !

Naming specification

1. The name of plasmids should start with letter "p".
2. Write "cut" on the tube if its contain has been digested with restriction enzymes. If there is enough space to write the restriction site, please do it.
3. Write the date on the bottle cap; write the concentration on the bottle.

Acknowledgements

SJTU-BioX-Shanghai

contact us

Contact us: sjtuigem@gmail.com or sjtuigem@gmail.com

Bio-X Institute, Shanghai Jiao Tong University, Dongchuan Rd. 800

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