Categories

FAQ

• What is the standard free cloning vector of BiOligo?

  (1) pUC series (pUC18/pUC19/pUC57), pBluescript II SK(+), PCR2.1, and similar vectors: contain several commonly used restriction endonuclease recognition sites.

  
  

  (2) pTG19-T vector: suitable for T/A cloning. Additionally, our company offers nearly 300 types of commercial expression vectors (such as pET series, pPIC series) for post-gene synthesis cloning and selection.

  
  

  If specific requirements exist, such as using particular or modified vectors, please provide the relevant information for these vectors to facilitate subsequent experiments promptly.

  
  

• Why might the plasmid received by the customer be difficult to cut or not fully cleave?

  Possible reasons are as follows:

  1.  Incorrect or absent enzyme recognition sequences on the plasmid.

  2.  Inappropriate conditions during the enzyme cleavage reaction.

  3.  Sensitivity of the restriction enzyme to DNA methylation.

  4.  Incorrect dilution or addition method of the enzyme.

  5.  High concentration of glycerol.

  6.  Partial or complete inactivation of the restriction enzyme.

  7.  Introduction of protective bases causing blockage at the enzyme cleavage sites.

  
  

  
  

  Solutions:

  1.  Check if the plasmid DNA contains DNA sequences recognized by the restriction enzyme.

  2.  Optimize the enzyme reaction system using the provided reaction buffer; increase enzyme concentration or use a fresh batch.

  3.  Check for DNA methylation and the enzyme's sensitivity to it.

  4.  Consider changing protective bases or perform PCR amplification of the target fragment and then enzymatic digestion.

  
  

• How to transport and store plasmids and their bacterial strains?

  We provide two tubes containing plasmids with completely accurate gene sequences (minimum of 3 μg). Plasmids can be transported at room temperature. After dissolution, they should be stored at -20°C and ideally avoid repeated freeze-thaw cycles.

• What are the requirements for sample submission when clients provide samples for services?

  For plasmid samples, they can be provided in the form of stab cultures, glycerol stocks, lyophilized plasmids, or plasmid solutions.

  1.  Stab Cultures: Please provide single colony stab cultures. Stab cultures ensure long-term preservation without a significant decrease in plasmid copy numbers. Place the sample in 1.5ml or 2ml Eppendorf tubes containing solid LB media supplemented with the corresponding antibiotic. Use a sterile toothpick to stab a single colony into the LB agar.

  
  

  2.  Glycerol Stocks: Add overnight grown bacterial culture to sterilized glycerol to achieve a final concentration of 20%. Store it in 1.5ml or 2ml Eppendorf tubes, ensuring proper sealing to avoid contamination.

  
  

  3.  Plasmid: Provide 3-5μg of plasmid dissolved in sterile, deionized water or in a dried form.

  
  

  4.  Note:

  a. Seal the Eppendorf tube containing the sample to prevent contamination or loss during transportation.

  b. Label the plasmid name and resistance on the tube.

  c. Provide additional information about the vector via email or attached note.

  
  

• Why might blunt-ended PCR products be challenging to clone?

  Since the typical PCR primers lack a phosphate group at the 5' end, the resulting PCR products also lack this phosphate group. When attempting cloning into dephosphorylated blunt vectors, they fail to integrate. Moreover, when cloned into non-dephosphorylated blunt vectors, the background noise is significantly high. In such cases, it's advisable to perform phosphorylation (PO4 modification) of the 5' ends of the PCR products.