Contribution

Document troubleshooting

Anyone who has done experiments knows that there will always be various problems during the experiment. However, these problems can generally be solved. Based on our own experimental experience and some data, we have compiled common problems and solutions in iGEM competition molecular cloning experiments.

We mainly talk about the use and maintenance of instruments and equipment in the lab (pipette, centrifuge, clean bench), and the problems that would happen during the process of PCR and agarose gel electrophoresis. We hope it can help freshmen carry out experiments better and faster at the beginning, which will provide a useful resource for future iGEM teams. We believe this troubleshooting can be a helpful resource for future iGEM teams.

Pipette

The pipette is a precision instrument for quantitatively pipetting micro-volume liquids in the laboratory. It can measure 0.01μL-10mL of liquid at a time and can achieve precise liquid configuration or transfer. It is an essential tool for molecular cloning experiments. Proper use of pipettes is the most basic experimental skill for carrying out molecular biology experiments.

1.Conventional usage of pipettes

1.1 Set the pipetting volume. The normal adjustment method is to adjust from the large range to the small range. Just rotate the scale counterclockwise. When adjusting from the small range to the large range, you should first adjust it to exceed the set volume scale and then adjust it back to the set volume. This can ensure that the pipette is accurate.

1.2 The scale of the pipette should be adjusted to the maximum after each experiment to allow the spring to return to its original shape to extend the service life of the pipette.

1.3 Select a pipette with a suitable measuring range. Do not use a large-capacity pipette to transfer small volumes of liquid to avoid affecting accuracy. At the same time, if you need to transfer a larger amount of liquid outside the measuring range, please use a pasteur pipette.

1.4 Assemble the pipette tip. Insert the pipette vertically into the tip, turn it half a turn left and right, and tighten it. It is very undesirable to hit the tip with the pipette. If you do this for a long time, the parts of the pipette will become loose due to the impact. In severe cases, the knob that adjusts the scale will get stuck.

1.5 Draw and drain the liquid. When aspirating liquid vertically, the tip of the tip should be immersed 3 mm below the liquid surface. Before aspirating, the pipette tip should be pre-rinsed in the liquid; suck slowly and release slowly. If the amount is small, the tip of the tip should be against the inner wall of the container.

1.6 The pipette filled with liquid should not be placed flat. The liquid in the tip of the pipette can easily contaminate the inside of the pipette and may cause the spring of the pipette to rust.

1.7 When drawing liquid, be sure to release your thumb slowly and steadily, and never let it go suddenly, to prevent the solution from being sucked in too fast and rushing into the liquid dispenser to corrode the plunger and cause air leakage. Or use a tip with a cartridge.

1.8 Liquids with high concentration and viscosity will produce errors. The compensation amount to eliminate the error can be determined by experiments.

1.9 When setting the measuring range, please pay attention to rotating it to the required measuring range. The numbers are clearly in the display window. The set measuring range is within the pipette measuring range. Do not turn the button out of the measuring range, otherwise, the mechanical device will be jammed and damaged.

1.10 After use, the pipette can be hung vertically in the pipette tip holder, but be careful not to drop it. Do not place the pipette horizontally or upside down when there is liquid in the pipette tip, as the liquid may flow back and corrode the piston spring.

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2.Unexplainable failure and solutions

Question 1: The pipette is leaking.

Possible reasons:

Solutions:

Question 2: There is always residue in the tips of the pipette.

Possible reasons:

Solutions:

Question 3: The action button is stuck or won't hold.

Possible reasons:

Solutions:

Question 4: The tip cannot be ejected or cannot be fixed.

Possible reasons:

Solutions:

Centrifuge

The centrifuge is the use of centrifugal force, separation of liquids and solid particles or liquids and liquids in the mixture of components of the machinery. Centrifuges are mainly used to separate solid particles from liquids in suspension, or to separate two liquids with different densities and immiscible in emulsion, and can also be used to exclude liquids from wet solids.

  1. 1.The centrifuge should always be in a horizontal position, the voltage of the external power supply system should be matched and a good grounding wire is required.
  2. The centrifuge in the pre-cooling state, the centrifuge cover must be closed, after centrifugation to dry the remaining water in the chamber, the centrifuge cover is open.
  3. In the centrifugation process, the operator shall not leave the centrifuge alone, once an abnormal situation occurs the operator can not turn off the power supply, to press STOP. Before the pre-cooling state, the centrifuge usage record should be filled out.
  4. Do not use shoddy centrifugal tube, do not use aging, deformation, or cracked centrifugal tubes. The quality of the centrifugal tube must be well-balanced and must be symmetrically placed in the casing to prevent vibration of the body.
  5. When starting the centrifuge, the top cover of the centrifuge should be covered before starting slowly. If the electric centrifuge has noise or body vibration, the power supply should be cut off immediately and troubleshooting should be done instantly.
  6. When centrifuging volatile or corrosive liquids, use a centrifuge tube with a cap and make sure the liquid does not leak out to avoid erosion of the chamber or causing accidents.
  7. Clean the machine chamber regularly.
  8. After separation, turn off the centrifuge first, and open the lid of the centrifuge only after the centrifuge stops rotating to take out the samples, and do not force it to stop moving with external force.
  9. When using the freezing centrifuge, in addition to paying attention to the above items, one should also pay attention to wiping the cavity of the action to be gentle, so as not to damage the temperature sensitivity of the cavity.

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Clean bench

Ultra-clean workbench (Clean Benches), in order to protect the experimental materials and design, through the fan will be the air into the pre-filter, through the static pressure box into the high-efficiency filter filtration, will be filtered air in the state of the vertical or horizontal airflow sent to the operating area to achieve the level of cleanliness of the hundred, to ensure that the production of the environmental cleanliness requirements.

  1. Before use, wipe the working surface with 75% alcohol, put the required items in the middle of the table on both sides of the working area, and sterilize the pipette tips, test tubes, Petri dishes, etc. before use.
  2. After 75% alcohol wiping, turn on the UV lamp for 15-20 minutes and irradiate the working area to sterilize and kill bacteria and viruses. In order to avoid too many items and lead to UV sterilization is not complete, the countertop usually only need to put pipettes, 75% alcohol spray bottle, 75% alcohol cotton, inoculators, alcohol lamps, markers and lighters.
  3. Turn off the UV after starting the ventilator, 10min before use, in order to exclude ozone and other harmful gases on the body.
  4. Turn the light switch on during operation.
  5. Scrub hands and forearms with 75% alcohol cotton before operation. Any items entering the ultra-clean table must be wiped with 75% alcohol before entering the ultra-clean table.
  6. Do not store unnecessary items on the work surface to keep the clean airflow in the work area undisturbed. Pass the fire before opening various bottle caps to fix the dust, pass the fire on the mouths of opened bottles and test tubes, and tweezers and inoculators should be cauterized by fire before use to remove bacteria again.
  7. Do not record on the workbench surface, and try to avoid actions that significantly disturb the airflow while working.
  8. After the work is completed, the table surface must also be wiped with 75% alcohol, and the used instruments and materials to the location, clean up the garbage, stop the fan running, turn off the lights, and take away the personal belongings that do not belong to the ultra-clean table surface.
  9. Operators should strictly follow the above procedures, when using pipettes, use alcohol cotton to wipe the pipette head. Pipette tips should be wrapped in newspaper and sterilized before use and should be for personal use only.

PCR

After the synthesized plasmid is obtained, plasmid PCR experiments are needed to verify whether the construction is successful. Plasmid verification PCR electrophoresis has no amplified bands, weak or mixed bands.

  1. The presence of complementary sequences or hairpin structures between primers and primers in the design of primers leads to the hybridization of electrophoresis. Too long primer or too high GC content leads to banding in electrophoresis.

    2. The solution: Firstly, Oligo or Primer is used to design primers containing target fragments of homologous arms of the carrier. The primers are designed in the conservative region, the length is generally 18-27bp, the upstream and downstream primers' Tm value is preferably 60-75℃, and the GC content is =40%-60%.

  2. Enzyme inactivation, contamination or lack of enzyme in the reaction system resulted in banding. Excessive enzyme levels lead to band dispersion.

    3. The solution: If TaqDNA polymerase is inactivated due to improper storage or transportation, a new enzyme or another enzyme from another source should be used to obtain satisfactory results. In case of band dispersion, the concentration of dNTP and MgCl2 should be reduced, and the amount of enzyme should be reduced.

  3. The template contains impurities, and the poor quality of the template may cause DNA depurination and affect the PCR result. Secondly, there are too many or too few templates.

    4. The solution: The plasmid extraction kit can be used for re-eluting and purification. The amount of template added should refer to the instructions of the system. The amount of plasmid DNA should be < 50ng, and the genome DNA should be < 200ng.

  4. The annealing temperature and denaturation and elongation time are not accurate; The temperature indicated by the PCR instrument is not consistent with the actual temperature, resulting in high temperature, and the enzyme is rapidly deactivated in the first few cycles; Or the annealing temperature is too low, the product mismatch or no PCR product, the denaturation time is short, the template denaturation is not complete; Elongation time is too short, amplification is not complete; Insufficient number of PCR cycles leads to weak bands.

    5. The solution: Selection of annealing temperature and determination of extension time for PCR. Reference should be made to primer length and GC content, as well as to fragment size and enzyme type. The gradient PCR reaction was designed to optimize the annealing temperature with 3℃ as the gradient. Set the extension time at 1kb/ min; Insufficient number of PCR cycles leads to weak bands, and the number of reaction cycles can be reasonably increased. At the same time, positive controls are added when DNA gel electrophoresis is performed to ensure that DNA gel and PCR procedures are problematic.

  5. The reaction buffer is not completely melted or sufficiently mixed. Improper use of the pipette leads to incorrect amount of system addition. For example, when the template is added, it is not inserted below the liquid level and then sucked and mixed, resulting in a small amount of template in the system or no template. No ice operation resulted in enzyme inactivation, and the gun head was not replaced in time, resulting in system contamination and thus heterozonation. The hot cover of the PCR instrument was not opened, resulting in evaporation of the system and failure of the reaction.

    6. The solution: Ensure that the reaction buffer melts completely and is thoroughly mixed; When the pipette is used for small volume pipette, it should be observed whether the liquid in the gun head exists and whether it is inserted below the liquid level for mixing; Early replication termination often occurs when using non-hot start polymerase, so the reaction system should be prepared or hot start polymerase should be used. When operating the PCR instrument, strictly follow the instructions and run after repeated inspection.

  6. The concentration difference between gel buffer and electrophoresis buffer is too large. The gel didn't set well; Agarose is of poor quality.

    7. The solution: Replace the electrophoretic buffer in time, pay attention to the gel dispensing system, select high-quality agarose, see the detailed solution below.

Agarose gel electrophoresis

Agarose gel electrophoresis is an important means to carry out molecular biology research, in this experiment often due to some operation is not standard and lead to the experimental results are not ideal, the following is a summary of some problems encountered in the experimental process and solutions:

  1. There are no bands in agarose gel electrophoresis.

    2. The solution: The most likely reason for this situation is that the nucleic acid dye has failed, or the addition of nucleic acid dye when the temperature of the agarose solution is too high, resulting in the failure of the nucleic acid dye, so that there is no band, excluding the first case should choose a new nucleic acid dye for comparative analysis, the second case should ensure that the agarose solution is cooled to about 60℃ before adding nucleic acid dye.

  2. The size of the glue hole is not uniform, resulting in uneven nucleic acid electrophoresis speed and wipeout phenomenon.

    3. The solution: This is due to insufficient agarose dissolution, or the gel time is too short, resulting in the nucleic acid glue is not fully cured, should be thoroughly dissolved in the preparation of nucleic acid glue to confirm that the solution is transparent, clear, oil-free, and before the sample to confirm that the nucleic acid glue is completely cured, generally 100ml 1% nucleic acid glue gel time is 30-40 min.

  3. The phenomenon of tailing or dispersion appeared in agarose gel electrophoresis.

    4. The solution: The first reason may be that too much sample loading causes the loading hole to be overloaded, under normal circumstances, a 5 mm wide comb can add 0.5 µg of nucleic acid. The amount of adding sample depends on the size of the adding hole and the size of the nucleic acid. If the amount of loading is too large, there will be a tailing or dispersion, and the amount of loading should be reduced. On the contrary, when the adding hole is large, the amount of loading sample should be increased accordingly, otherwise the strip will be shallow or even unclear. The second reason may be that the voltage is too large, which causes the strip to bend and tail. At this time, the electrophoretic voltage should be reduced, the recommended voltage is 5-10 V/cm, generally 110-130 V, and the maximum is not more than 150 V.

Reference:

[1] GUIDE TO PIPETTING. (https://pipette.com/guide-to-pipetting.html)

[2] Simatos D, Jacobs IE, Dobryden I, et al. Effects of Processing-Induced Contamination on Organic Electronic Devices. Small Methods. 2023, e2300476. doi:10.1002/smtd.202300476.

[3] Beroz J, Hart AJ. Universal handheld micropipette. Rev Sci Instrum. 2016, 87(11):115112. doi:10.1063/1.4966989.

[4] Bond EW. Preparing Graduated Pipettes. Can J Comp Med Vet Sci. 1945, 9(6):166-167.

[5] GREEN GF. A portable micro-centrifuge. J Clin Pathol. 1963, 16(1):90. doi:10.1136/jcp.16.1.90.

[6] Beams HW, Kessel RG. Development of centrifuges and their use in the study of living cells. Int Rev Cytol. 1987, 100:15-48. doi: 10.1016/s0074-7696(08)61697-6.

[7] Coté RJ. Aseptic technique for cell culture. Curr Protoc Cell Biol. 2001, Chapter 1. doi:10.1002/0471143030.cb0103s00.