Q: No colony or less colony

A: 

1. Primer design error: Confirm that the primer sequences contain a 15 bp homologous sequence identical to the insertion region of the vector. 

2. Poor purity of the insert fragment: Purify both the vector and insert fragment using gel extraction, and proceed with ligation only after confirming a single band on agarose gel electrophoresis of the gel-extracted products. 

3. Low transformation efficiency: Use freshly prepared competent cells or highly efficient commercial competent cells. 

4. Poor ratio of linearized vector or insert fragment within the reaction system: Calculate the amounts of each component according to the manual, and assess sample quality and concentration using agarose gel electrophoresis. 

Q: High false-positive cloning rate

A: 

1. Incomplete linearization of cloning vector may lead to higher false positives. It is recommended to increase the amount of restriction enzyme used, extend the reaction time, and purify the digested products using gel extraction. 

2. Contamination with plasmids carrying the same resistance gene. Pay attention to the cleanliness of the experimental environment and eliminate background plasmids. 

3. Loss of antibiotic resistance in transformation plates. It is recommended to use freshly prepared plates containing the correct and appropriate concentration of antibiotics.

Q: Incorrect insert in clones

A: 

Optimize the PCR system to improve specificity and use gel extraction to purify PCR products. High-quality products provide reliable support for experiments. You deserve to have TransGen seamless cloning products!

Q：Unusual pH in medium.

A：

· Possible Cause: Incorrect set up for carbon dioxide (CO2) pressure. Suggested Solution: Increase or decrease CO2 pressure in the incubator based on concentration of sodium bicarbonate in medium.

· Possible Cause: No carbon dioxide (CO2) exists in the incubator. Suggested Solution: Monitor carbon dioxide (CO2) pressure and change CO2 tank timely.

· Possible Cause: Overly tight caps on cell culture flasks. Suggested Solution: Loosen caps one-quarter turn or use culture flask with vented cap.

· Possible Cause: Bacterial or fungal contamination. Suggested Solution: Discard cells and the medium.

· Possible Cause: Incorrect balanced salts in medium. Suggested Solution: Use Earle’s salts-based medium in CO2 environment and Hanks’ salts-based medium in atmospheric conditions.

· Possible Cause: Bicarbonate buffer system provides insufficient buffering. Suggested Solution: Add HEPES buffer to attain a final concentration of 10 to 25 mM.

A：

· Possible Cause: Change happens in medium or serum. Suggested Solution: Compare glucose, amino acids, and other components in different media; Increase cell inoculation density; Transfer cells to fresh medium.

· Possible Cause: Lack, depletion or degradation of components essential for cell growth (such as L-Glutamine and growth factors). Suggested Solution: Use fresh medium or add essential components to the medium.

· Possible Cause: The initial cell inoculation density is too low. Suggested Solution: Increase cell inoculation density.

· Possible Cause: Mycoplasma contamination. Suggested Solution: If the culture has been detected to have mycoplasma contamination, discard the culture or use mycoplasma elimination reagent.

· Possible Cause: Cells have been passaged too many times. Suggested Solution: Use new cells.

· Possible Cause: Low levels of bacterial or fungal contamination. Suggested Solution: Use antibiotic medium; If the culture is contaminated, discard it.

· Possible Cause: Medium or serum is stored incorrectly. Suggested Solution: Serum should be stored at -5 to -20 ℃. Medium should be stored at 2-8 ℃ in dark. Complete Medium should be stored at 2-8 ℃ in dark and used within 2 weeks.

A：

· Possible Cause: Cells are overly trypsinized. Suggested Solution: Reduce trypsinization time or trypsin concentration.

· Possible Cause: Poor cell state. Suggested Solution: Use new cells.

· Possible Cause: Mycoplasma contamination. Suggested Solution: Discard the culture if it has been detected to have mycoplasma contamination, or use mycoplasma elimination reagent.

· Possible Cause: No attachment factors in medium. Suggested Solution: For serum-free culture, be sure attachment factors are contained or use coated culture vessels.

· Possible Cause: No carbon dioxide (CO2) exists in the incubator. Suggested Solution: Monitor carbon dioxide (CO2) pressure and change the CO2 tank timely.

· Possible Cause: Temperature fluctuation in the incubator. Suggested Solution: Monitor temperature in the incubator.

· Possible Cause: Too high toxicity of transfection reagents; Too high passage number of cells; Low purity of plasmids. Suggested Solution: Use transfection reagents of low toxicity or change medium timely after transfection; Use cells of low passage number; Use plasmids of high quality.

· Possible Cause: Big cell damage during thawing or cryopreservation. Suggested Solution: Use new cells.

· Possible Cause: Incorrect osmotic pressure of medium. Suggested Solution: Check the osmotic pressure of complete medium. Most mammalian cells can tolerate osmotic pressure of 260-350 mOsmol/kg and most insect cells can tolerate osmotic pressure of 340-380 mOsmol/kg.

·Possible Cause: Excessive toxic metabolites accumulate in the medium. Suggested Solution: Change fresh medium.

Q: Low yield

A:

· Check if the cell growth and its culture conditions are suitable for plasmid amplification. Selecting high-copy number plasmids is recommended.

· If the copy number of the plasmid is low, increase the amount of collected bacteria appropriately, but ensure that the bacteria can be completely lysed.

· If the lysate is too sticky, reduce the amount of the bacteria appropriately.

· Too long culturing time or storage time at 2-8 oC can significantly reduce yield, so it is recommended to use fresh bacteria.

Q: Genomic DNA contamination

A: 

The lysis time should not exceed 5 minutes.

Q: Downstream restriction enzyme cleavage affected

A: 

After using wash buffer, remove the residual liquid by centrifugation as much as possible. Add elution buffer after the silica membrane has been dried completely.

Q: Low RNA Yield 

A: 

• Low RNA amount in the Sample: For samples with low RNA amount, such as fibrous tissues (e.g., muscle tissue or plant tissues with high cellulose content), increase the starting material proportionally. 

• High Protein and Lipid Content in the Sample: For samples with high protein or lipid content, perform an additional chloroform extraction on the supernatant to improve RNA recovery. 

• Inappropriate RNA Precipitation Conditions: When precipitating RNA with isopropanol, ensure a strict 1:1 ratio of isopropanol to the extraction solution. Deviations can significantly affect RNA precipitation efficiency and purity. For challenging samples (e.g., those containing polysaccharides or polyphenols), adding an appropriate amount of sodium citrate can enhance precipitation efficiency.

Q: Genomic DNA contamination in RNA

A:

• High nucleic acid content in the material: The nucleic acid content is high in tissues such as spleen and thymus, and multiple phenol/chloroform extractions can be performed to remove excess DNA. DNase I can also be added after extraction to degrade DNA.

• Excessive material: Increase the amount of reagents.

•Inappropriate RNA precipitation conditions: When using isopropanol to precipitate RNA, the ratio of isopropanol added to the extract is too high, and the pH value of the solution is too low, which can easily cause DNA to precipita.

Q: The elution solution turns black or the silica membrane becomes discolored (for animal tissues or mouse tail materials)

A:

• Pigments from the tissues bind heavily to the spin column and are eluted along with the DNA:

Ensure ethanol has been added before passing through the column, as ethanol prevents pigments from binding to the silica membrane. During the lysis and binding step, increase the centrifugation speed and extend the centrifugation time if necessary.

• RNA contamination: Add an appropriate amount of RNase A during the sample preparation process to degrade RNA.

Q: Issues affecting downstream enzymatic digestion

A:

• Residual ethanol in the purified DNA: Residual ethanol might be carried over during the washing step. Thoroughly dry the spin column by centrifuging it at maximum speed for 2–3 minutes before adding the elution buffer.

• Residual salts in the purified DNA: Follow the correct washing procedure. After washing with different CB solutions, ensure to perform a final wash with WB solution.

Q: Unexpected cleavage bands

A:

Determine which are the correct bands: Detect the digested products and the control DNA by electrophoresis, and then determine if the bands come from star activity or incomplete digestion.

Contamination of the DNA sample: Reprepare or purify samples.

Other recognition sites included: Check DNA sequences.

Q：Why are the bands blurred?

A：

· Too high voltage or too long running time produces too much heat which increases temperature of the buffer. Please refer to product manual.

· Buffer is old. Use fresh solutions and pre-chill the buffer before use to get a great result.

· Gel concentration is low. Try proper gel concentration.

· Gel is old. Use newly made gels.

A：

· It is suggested to load protein marker to the middle lane because bands at the edges of the gel may become wider or distorted due to edge effects or ionic strength difference.

· The gel is uneven or contains bubbles. It is also possible that some gel residues exist in the sample well.

A：

 Mostly, gel concentration is not suitable, which should be within recommended concentration range. Sometimes buffer and gel are not fresh enough to lead to a good electrophoresis result.

A：

· The transfer sandwich should be prepared properly. Otherwise bands may become missed or faint in protein transfer.

· Transfer condition needs to be optimized. 200 mA of 2 hours or 100 mA overnight is recommended.

A：

The recommended methanol concentration is 15%. Too high concentration may cause protein passing through the membrane and finally transferred to the filter paper.

A：

There is no need to prepare ECL mix in dark, but it is necessary to use fresh ECL mix and continue the following steps in the dark room immediately after ECL mix is prepared well.

A：

Light emission can last up to 2 hours. It is more intense during the first 30 minutes but decreases with time. Longer exposure times may be necessary as time elapses.

A：

Exposure time or developing time is too long.

A：

It is due to too much sample loaded or too long exposure time. It is also possibly because of too high concentration, low specificity or low purity of secondary antibodies.

Q：Chromatographic column is blocked.

A：

Protein samples may contain solid impurities. Mostly, it’s because bacterial lysates are loaded directly. It is recommended to process protein by microfiltration.

A：

· His tag is not present, which is possibly due to incorrect vector construction, premature termination of expression (His tag on C-terminal), the internal ribosome entry site (His tag on N-terminal), etc. It is recommended to sequence or change the expression vector.

· His Tag is inaccessible folded and buried inside the protein's three-dimensional conformation, which happens mostly when denatured inclusion bodies are purified. It is recommended to fully denature protein by adding stronger denaturants (Use 6 M Guanidine hydrochloride to replace 8 M urea), Cosolvents (SDS) or reducing agents (DTT orβ- Mercaptoethanol).

·  Buffer composition is incorrect. When Ni-NTA resin or Ni-IDA resin were used to purify His tag protein, some components in the sample buffer and equilibration buffer may interfere with the binding of protein.

Some recommended concentrations:

EDTA: ≤0.1 mM (Not Recommended to use);

DTT: ≤1 mM (Not Recommended to use);

β- Mercaptoethanol: ≤20 mM (Use cautiously);

Imidazole: ≤20 mM (Protein dependent).

A：

·  Elution conditions are not optimal. It is recommended to apply Imidazole or pH gradient elutions. The optimal elution condition varies from protein to protein, which needs to be optimized step by step.

·  Contaminant proteins non-specifically bind to the purification resin. Add Imidazole (The recommended concentration is ≤ 20 mM which depends on protein) to sample buffers and equilibration buffers to inhibit non-specific bindings.

·  Contaminant proteins non-specifically bind to the target protein. Add non-Ionic detergents (Triton X-100: ≤1%), Glycerol (≤10%), NaCl (≥300 mM), reducing agents (β- Mercaptoethanol: ≤20 mM) or some other components to sample buffers and equilibration buffers.

·  Protein has been degraded. It is recommended to purify protein at low temperature and add protease inhibitors (such as PMSF) into sample buffers and equilibration buffers.

·  Premature termination of expression (His tag on C-terminal) or internal ribosome entry site (His tag on N-terminal) is present. It is recommended to change the expression vector.

A：

·  Protein content is too low. It is recommended to replace Coomassie Blue Staining with detection methods with higher sensitivity, such as Silver Staining and Western Blot. Alternatively, optimize upstream conditions of protein expression to obtain more protein.

·  Protein does not bind to the resin. It is recommended to detect protein content of the sample, the flow-through fraction and the wash fraction.

·  Protein binds to the resin too strongly. It is recommended to enhance elution strength using higher concentration of Imidazole or lower pH.

Q：Low or no protein expression

A：

Choose correct expression vectors and strains.

· T7 promoter-based expression vectors (like pET vector series) should be paired with strains like BL21 (DE3), BL21 (DE3) pLysS and Transetta (DE3). For non-T7 promoter-based expression vectors (e.g. pGEX and pMAL vector series with Tac promoter) should be paired with the BL21 expression strain.

· For toxic proteins, it is recommended to use strains with low background expression and strict regulation such as the BL21 (DE3) pLys S strain.

· For proteins with rare codons or proteins from eukaryotic genes, strains such as Transetta (DE3) are recommended.

2, Try different expression vectors and strains Different proteins have different preferences for vectors and strains. If protein expression cannot be significantly improved by optimizing induction expression conditions, it is recommended to change strains or expression vectors.

3, Optimization of expression conditions

· Choose different media. For some proteins, a proper amount of glucose (0.1%-0.5%) addition can significantly increase the amount of expression.

· Higher temperature, higher inducer concentration, and longer expression time can generally speed up expression and promote the accumulation of target proteins, and thereby increase the amount of expression. However, the solubility may be reduced leading to the formation of inclusion bodies.

· The growth status of cells has a significant impact on protein expression and can be monitored by measuring the OD600 value of the bacteria solution. For most proteins, induction should be performed in llogarithmic growth phase (OD600=0.5) of the strain.

Q：Target proteins are insoluble with the formation of inclusion bodies

A：

Firstly, confirm whether the target protein has been expressed.

· After the lysis of the bacteria and centrifugation, confirm whether the target protein has been expressed and whether the inclusion body has been formed by detection of whole bacteria, supernatant and precipitation.

· The formation of the inclusion body is closely related to the protein's structure, expression system, induced expression conditions and other factors. In the case that the protein's structure cannot be changed, different expression vectors or strains can be tried to enhance protein solubility and optimize for an expression system that is suitable for a specific protein.

· It is currently acknowledged that the formation of the inclusion body is due to too rapid protein accumulation in the cell, thus the protein cannot be correctly folded resulting in protein aggregation and precipitation. The accumulation of proteins can be slowed down by optimizing induced expression conditions, such as reducing induction temperature, reducing the concentration of the inducer, shortening expression time and reducing the OD value of the bacteria solution in induction.

Q： Incorrect target protein size

A：

· Determining molecular weight is affected by the structure of the protein. The molecular weight can be accurately determined by heating the protein to denatured form.

· Confirm if protein expression is complete and early termination has occurred.

· If dimers or even multimers are formed, the molecular weight can be accurately determined by heating the protein to denatured form, breaking disulfide bonds (Add DTT, β-ME or other reducing agents) or other methods that can destroy the secondary structure of the protein.

Q：How to choose the right cloning vector?

A：

For Taq-amplified PCR products, choose T cloning vector. For Pfu-amplified PCR products, choose Blunt cloning vectors, or T cloning vector after adding a single deoxyadenosine (A) (the result will be compromised compared with ligating Blunt vectors directly). For plasmid templates, dual antibiotic resistance vectors are recommended.

Q： What is the appropriate amount of inserted fragments?

A：

· The amount of fragment addition can be adjusted according to the length of the fragment. The optimal vector-to-insert molar ratio is 1:7.

· It can be roughly calculated at the ratio of "1 kb- to-20 ng" (e. g. 20ng for 1 kb inserted DNA, ; 30 ng for 1.5 kb inserted DNA) (The amount can be quantified roughly by TransGen's DNA molecular weight standard on the gel.)

· The maximum amount of fragment addition is 4 μl, and do not exceed this limit even if the fragment concentration is very low. The minimum amount of fragment addition is 0.5 μl, which can be replenished with sterile water for easy operation.

· Reaction temperatures range: 20°C to 37°C . The optimal reaction temperature is 25°C.

Q：How long should the reaction last?

A：

· Reactions for most PCR products within 1 kb can be completed in 5 minutes under room temperature.

· There are several situations in which reaction time can be extended for better results:

1. Inserts with special structures (high AT content/ high GC content/ inverted repeat sequence): 10-20 minutes.

2. Inserts from gel extraction or A overhangs addition: 10-15 minutes (this condition applies to fragments up to 3 kb).

3. PCR products larger than 3 kb: 15-20 minutes.

Q：How long can the ligated mixture be stored?

A：

The ligated mixture can be stored in a refrigerator of -20°C or 2-8°C, and should be used within one week.

Q：Unable to cleave the restriction sites in the multiple cloning site.

A：

The screened colonies come from the plasmid templates rather than the ligation mixture. It is recommended to use double antibiotic-resistant vectors and choose the resistance that plasmid templates don't contain for screening.

Q：Few colonies or low cloning efficiency? 

A：

There are many factors that affect the number of colonies and cloning efficiency, such as the transformation efficiency of competent cells, the quality of the inserts, the structure of the gene, the length of the inserts, insert-to-vector molar ratio, etc. If you find that the number of colonies is low or the cloning is inefficient, you can try the following methods:

· The transformation efficiency of competent cells has significant influence on the number of colonies, and their relationship is not linear. For example, when the transformation efficiency of the competent cells is 1 x 109 cfu /µg DNA, the number of colonies is 1000. But when the transformation efficiency is 1 x 108 cfu / µg DNA, the number of colonies is less than 100. In order to ensure a high number of colonies, please choose competent cells Trans1-T1 with high transformation efficiency.

· Use fresh PCR products: PCR products should be stored at 2-8℃ and used within 1-2 days. For isolated DNA from gel extraction, the UV light exposure time should be minimized and the reaction time should be extended appropriately (10-15 minutes).

· When the concentration of the target fragment is very low, concentrate to ensure more chances of molecular collision during reaction.

· Toxic genes: Use Trans10 competent cells.

· Genes with special structures (high AT content/ high GC content/ inverted repeat sequence): extend reaction time to 10-20 minutes.

Q：Failure to verify the transformants by PCR？

A：

When expected amplified products, or vector self-ligation have not been observed by PCR with universal primers, this means PCR has failed. Optimize the PCR reaction conditions again or extract the plasmids which can be used as templates for amplification or digested by restriction enzyme to identify colonies containing transformants.

Q：Colonies show light blue or "Fish eye"

A：

If the insert does not affect the LacZ reading frame, or the insert is too short, the colonies appear light blue or "Fish eye", and can still be verified as usual.